Genetic engineering of non-human animals for the production of chimeric antibodies

ABSTRACT

The invention provides non-human cells and mammals having a genome encoding chimeric antibodies and methods of producing transgenic cells and mammals. Certain aspects of the invention include chimeric antibodies, humanized antibodies, pharmaceutical compositions and kits. Certain aspects of the invention also relate to diagnostic and treatment methods using the antibodies of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation Application U.S. patent applicationSer. No. 15/463,761 (allowed), filed on Mar. 20, 2017, which is adivisional of U.S. patent application Ser. No. 15/408,114 filed on Jan.17, 2017, now U.S. Pat. No. 10,494,445 issued on Dec. 3, 2019, which isa divisional of U.S. patent application Ser. No. 13/638,522 filed onMar. 5, 2013, now U.S. Pat. No. 9,580,491 issued on Feb. 28, 2017, whichis a 371 National Phase Application of PCT/US2011/030823 filed on Mar.31, 2011, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 61/319,690 filed Mar. 31, 2010 andU.S. Provisional Patent Application No. 61/361,302 filed Jul. 2, 2010.The content of each of these patent applications is incorporated byreference in its entirety.

BACKGROUND Technical Field

The present invention is directed generally to chimeric immunoglobulinchains, antibodies and non-human animals and cells, and the productionthereof.

Description of the Related Art

Disease therapies utilizing monoclonal antibodies (mAbs) haverevolutionized medicine, and mAb-based drugs are now utilized in thetreatment of cancer, autoimmunity, inflammation, macular degeneration,infections, etc. However, the available technologies for generation anddiscovery of mAbs for use in the prevention and treatment of diseasesand disorders have significant drawbacks including inefficiency, absenceor loss of sufficient potency, absence or loss of specificity and theinduction of an immune response against the therapeutic mAb. The firstattempts to use mAbs as therapeutics were hindered by the immunogenicityof the mouse amino acid composition of the mAbs. When administered tohumans, the mouse amino acid sequence elicited a human anti-mouseantibody (HAMA) response that dramatically reduced the potency andpharmacokinetics of the drug as well as causing severe and potentiallyfatal allergic reactions.

Additional methods to generate mAb therapeutics include chimerized mAbs(cmAbs) created through recombinant DNA technology combining amouse-derived variable domain appended to a human constant region. Othermethods of generating antibodies involve humanizing mAbs in vitro tofurther reduce the amount of mouse amino acid sequence in a therapeuticmAb. Antibody-display technologies developed to generate “fully-human”antibodies in vitro have yet to adequately mimic the natural antibodymaturation process that occurs during an in vivo immune response (seepg. 1122-23, Lonberg, Nat. Biotech. (2005) 23:1117-1125.) mAbs developedusing these methods can elicit an immune response that can reduceefficacy and/or be life-threatening, and these processes are typicallytime-consuming and costly. Also, during the molecular processes inherentin these methods, loss of affinity and epitope shifting can occur,thereby reducing potency and introducing undesirable changes inspecificity.

Transgenic mice have been engineered to produce fully human antibodiesby introducing human antibody transgenes to functionally replaceinactivated mouse immunoglobulin (Ig) loci. However, many of thesetransgenic mouse models lack important components in the antibodydevelopment process, such as sufficient diversity in the genes fromwhich antibody variable regions are generated, the ability to make IgD(Loset et al., J. Immunol., (2004) 172:2925-2934), important cisregulatory elements important for class switch recombination (CSR), or afully functional 3′ locus control region (LCR) (e.g., U.S. Pat. No.7,049,426; and Pan et al., Eur. J. Immunol. (2000) 30:1019-1029). Sometransgenic mice contain yeast artificial chromosomes or human minilocias integrated transgenes. Others carry transchromosomes that exhibitvarious frequencies of mitotic and meiotic instability. Furthermore, thefully human constant regions of these transgenic mice functionsub-optimally due to reduced activity in conjunction with otherendogenous and trans-acting components as compared to wild-type mice,e.g., the BCR signal transduction apparatus, (Igα and Igβ) and Fcreceptors (FcR), respectively.

Knock-in mice have also been genetically engineered to produce chimericantibodies that are composed of human V domains appended to mouse Cdomains that remain fully intact, with the fully-intact portionscomprising all genomic DNA downstream of the J gene cluster (see U.S.Pat. Nos. 5,770,429 and 6,596,541 and U.S. Patent ApplicationPublication No. 2007/0061900). Human V regions from these mice can berecovered and appended to human constant region genes by molecularbiological methods and expressed by recombinant methods to producefully-human antibodies. The antibodies from these mice may exhibitreduction or loss of activity, potency, solubility etc. when the human Vregion is removed from the context of the mouse C domains with which itwas evolved and then appended to a human C region to make a fully humanantibody. Furthermore, because of the unique and differing structures ofthe mouse immunoglobulin lambda locus versus that of the humanimmunoglobulin lambda locus and because the endogenous 3′ enhancer ofthe mouse lambda locus may be defective, the described knock-in approachwould be expected to yield an inefficiently functioning lambda locus.

Methods of transgene DNA construction for introduction into eukaryotic,particularly metazoan, species have employed DNA isolated from genomiclibraries made from isolated natural DNA. Engineering of the clonednatural DNA into the final desired design for a transgene is typicallyachieved through processes of recombination that are cumbersome,inefficient, slow and error-prone and constrained by the availability ofthe DNAs present in genomic libraries. In some instances, it is desirousto construct a transgene from an organism, strain or specific haplotypethereof for which a genomic library is not readily available but forwhich either partial genomic sequence or transcriptome sequenceinformation is available. These hindrances prevent the creation oftransgenes comprising complexly reconfigured sequences and/or transgenesdesigned to comprise chimeric DNA sequence from different species ordifferent strains or different haplotypes of the same species. As aconsequence, the engineering of highly-tailored transgenes foreukaryotes, particularly metazoans, is prevented.

Current methods of developing a therapeutic mAb can alter functions ofthe antibody, such as solubility, potency and antigen specificity, whichwere selected for during initial stages development. In addition, mAbsgenerated by current methods have the potential to elicit a dangerousimmune response upon administration. Current human and chimeric antibodyproducing mice lack appropriate genetic content to function properly,e.g., genetic diversity, cis regulatory elements, trans actingregulatory elements, signaling domains, genetic stability. It would bebeneficial to develop methods and compositions for the enhancedgeneration and discovery of therapeutic antibodies and that retainpotency and specificity through the antibody generation, discovery,development, and production process without eliciting an immuneresponse, as well as methods of producing such antibodies. Some of thetransgene compositions comprise DNA sequences so complexly modified thatconstruction of these improvements and derivation of products therefromhave been prevented. While mice are preferred because of their economyand established utility, a broad solution across multiple species isdesirable. The present invention provides a solution for making andintroducing such transgenes, improving the genetic background into whichthese transgenes would function if deployed in a mouse, and, inparticular instances, generating improved antibodies in transgenicanimals.

BRIEF SUMMARY

The present invention relates to non-human animals and cells,transgenes, antibodies, methods, compositions, including pharmaceuticalcompositions, as well as kits of various embodiments disclosed herein.More specifically, the present invention relates to methods,compositions and kits relating to chimeric Ig chains and antibodiesproduced by the non-human animals and cells and the human antibodies andfragments thereof engineered from the variable domains of said chimericantibodies. In certain embodiments of the invention, the non-humananimals are mammals.

One embodiment of the invention relates to a method of producing a cellcomprising a genome that comprises a chimeric immunoglobulin chain,wherein the immunoglobulin chain comprises a non-endogenous variabledomain and a chimeric constant region, comprising the steps of (1)designing a DNA construct in silico, wherein said construct comprisesone or more non-endogenous V, (D) and/or J gene segments and one or morenon-endogenous constant region gene segments; (2) producing said DNAconstruct; and (3) introducing the construct into the genome of a cell.In certain embodiments, the non-endogenous variable domain is human. Inanother embodiment, the chimeric constant region comprises a mouseconstant domain gene segment. In one embodiment, the chimeric constantregion is encoded by a non-endogenous polynucleotide sequence derivedfrom two or more non-endogenous species, alleles and/or haplotypes. Inyet another embodiment, the non-endogenous variable domain is encoded bya polynucleotide sequence derived from two or more species, allelesand/or haplotypes. In certain embodiments, the chimeric immunoglobulinchain is a light chain.

In certain other embodiments, the chimeric immunoglobulin chain is aheavy chain. In a related embodiment, the chimeric constant regioncomprises a non-endogenous CH1 domain. In another related embodiment,the method further comprises the steps of designing a second DNAconstruct in silico, wherein said construct comprises a non-endogenousimmunoglobulin light chain; producing said second DNA construct; andintroducing the second construct into the genome of a cell. In oneembodiment, the non-endogenous light chain comprises one or more humanVκ gene segments. In another embodiment, the non-endogenous light chainfurther comprises one or more human Jκ and Cκ gene segments. In yetanother embodiment, the non-endogenous light chain comprises 8 or morehuman Vλ gene segments. In a related embodiment, the non-endogenouslight chain further comprises 7 or more human Jλ-Cλ gene segment pairs.

One embodiment relates to a non-human cell comprising a genome thatcomprises a chimeric immunoglobulin chain, wherein the immunoglobulinchain comprises a non-endogenous variable domain and a chimeric constantregion, wherein the cell is produced by a method comprising the steps of(1) designing a DNA construct in silico, wherein said constructcomprises one or more non-endogenous V, (D) and/or J gene segments andone or more non-endogenous constant region gene segments; (2) producingsaid DNA construct; and (3) introducing the construct into the genome ofa cell. Another embodiment encompasses a non-human animal generated fromthe cell. Another embodiment provides a chimeric immunoglobulin heavychain produced by the non-human animal. Certain embodiments provide achimeric antibody produced by the non-human animal.

Another embodiment of the invention provides a chimeric immunoglobulinheavy chain comprising a non-endogenous variable domain and a chimericconstant region, wherein the non-endogenous variable domain is derivedfrom a non-human animal. In a related embodiment, the chimeric constantregion comprises a non-endogenous CH1 domain. One embodiment provides achimeric immunoglobulin heavy chain comprising a non-endogenous variabledomain and a chimeric constant region, wherein the chimeric constantregion is encoded by a non-endogenous polynucleotide sequence derivedfrom two or more non-endogenous species, alleles and/or haplotypes.Another embodiment provides a chimeric immunoglobulin heavy chaincomprising a non-endogenous variable domain and a chimeric constantregion, wherein said non-endogenous variable domain is encoded by apolynucleotide sequence derived from two or more species, alleles and/orhaplotypes.

Yet another embodiment is directed to a polynucleotide encoding thedisclosed chimeric immunoglobulin heavy chain. In particularembodiments, the polynucleotide comprises coding and non-codingsequences. In certain embodiments, the polynucleotide is synthetic. Oneembodiment relates to a construct comprising the polynucleotide apolynucleotide encoding the disclosed chimeric immunoglobulin heavychain.

Another embodiment of the invention provides a chimeric antibody, or anantigen-binding fragment thereof, comprising (1) a chimericimmunoglobulin heavy chain, wherein the chimeric heavy chain comprises anon-endogenous heavy chain variable domain and a chimeric heavy chainconstant region, and (2) a non-endogenous immunoglobulin light chain,wherein the chimeric heavy chain constant region is derived from two ormore non-endogenous species, alleles and/or haplotypes. Yet anotherembodiment provides a chimeric antibody, or an antigen-binding fragmentthereof, comprising (1) a chimeric immunoglobulin heavy chain, whereinthe chimeric heavy chain comprises a non-endogenous heavy chain variabledomain and a chimeric heavy chain constant region, and (2) anon-endogenous immunoglobulin light chain, and wherein saidnon-endogenous heavy chain variable domain is derived from two or morespecies, alleles and/or haplotypes. One embodiment relates to a chimericantibody, or an antigen-binding fragment thereof, comprising a chimericimmunoglobulin heavy chain, wherein the chimeric heavy chain comprises anon-endogenous variable domain and a chimeric constant region, andwherein the variable domain is derived from a non-human animal. In arelated embodiment, the disclosed chimeric antibody, or antigen-bindingfragment thereof, further comprises a non-endogenous light chain.

One embodiment of the invention provides a non-human cell comprising agenome that comprises a chimeric immunoglobulin heavy chain comprising anon-endogenous variable domain and a chimeric constant region, whereinthe non-endogenous variable domain is derived from a non-human animal.In a related embodiment, the genome of the cell further comprises anon-endogenous immunoglobulin light chain. In particular embodiments,the genome of the cell comprises a non-endogenous Igκ light chain and anon-endogenous Igλ light chain. In certain embodiments, the cellcomprises an inactivated endogenous immunoglobulin locus. One embodimentprovides a chimeric antibody produced by the disclosed cell.

Yet another embodiment provides a non-human cell comprising a genomethat comprises a chimeric immunoglobulin heavy chain comprising anon-endogenous variable domain and a chimeric constant region, whereinthe constant region is derived from two or more non-endogenous species,alleles and/or haplotypes. One embodiment provides a non-human cellcomprising a genome that comprises a chimeric immunoglobulin heavy chaincomprising a non-endogenous variable domain and a chimeric constantregion, wherein the non-endogenous variable domain is derived from twoor more species, alleles and/or haplotypes. Another embodiment providesa non-human cell comprising a genome that comprises a synthetictransgene encoding a chimeric antibody, or an antigen-binding fragmentthereof, comprising (1) a chimeric immunoglobulin heavy chain, whereinsaid chimeric heavy chain comprises a non-endogenous heavy chainvariable domain and a chimeric heavy chain constant region. In certainembodiments, the genome of the disclosed cell further comprises anon-endogenous immunoglobulin light chain. In one embodiment, the genomeof the cell comprises a non-endogenous Igκ light chain and anon-endogenous Igλ light chain. In particular embodiments, the cellcomprises an inactivated endogenous immunoglobulin locus. Anotherembodiment provides for a chimeric antibody produced by the cell.

Another embodiment of the invention relates to a non-human animalcomprising a genome that comprises a chimeric immunoglobulin heavy chaincomprising a non-endogenous variable domain and a chimeric constantregion, wherein the non-endogenous variable domain is derived from anon-human animal. In a related embodiment, the genome of the animalfurther comprises a polynucleotide sequence encoding a non-endogenousimmunoglobulin light chain. In certain embodiments, the genome of theanimal comprises a non-endogenous Igκ light chain and a non-endogenousIgλ light chain. In another embodiment, the animal comprises aninactivated endogenous immunoglobulin locus. In certain embodiments, theanimal is a mouse. Another embodiment provides a chimeric antibodyproduced by the non-human animal.

Yet another embodiment of the invention provides a non-human animalcomprising a genome that comprises (1) a chimeric immunoglobulin heavychain, wherein the chimeric heavy chain comprises a non-endogenous heavychain variable domain and a chimeric heavy chain constant region, and(2) a non-endogenous immunoglobulin light chain, wherein the chimericheavy chain constant region is derived from two or more non-endogenousspecies, alleles and/or haplotypes. Another embodiment provides anon-human animal comprising a genome that comprises (1) a chimericimmunoglobulin heavy chain, wherein the chimeric heavy chain comprises anon-endogenous heavy chain variable domain and a chimeric heavy chainconstant region, and (2) a non-endogenous immunoglobulin light chain,wherein the non-endogenous heavy chain variable domain is derived fromtwo or more species, alleles and/or haplotypes. One embodiment providesa non-human animal comprising a genome that comprises a synthetictransgene encoding a chimeric antibody, or an antigen-binding fragmentthereof, comprising (1) a chimeric immunoglobulin heavy chain, whereinthe chimeric heavy chain comprises a non-endogenous heavy chain variabledomain and a chimeric heavy chain constant region. In particularembodiments, the genome further comprises a non-endogenousimmunoglobulin light chain. In certain embodiments, the genome of theanimal comprises a non-endogenous Igκ light chain and a non-endogenousIgλ light chain. In particular embodiments, the cell comprises aninactivated endogenous immunoglobulin locus. Another embodiment providesa chimeric antibody produced by the disclosed animal.

One embodiment of the invention provides a non-human animal comprisingan inactivated endogenous Ig locus, wherein the endogenous Ig locuscomprises a deletion that impairs formation of a functional variabledomain and formation of a constant region capable of driving primary Bcell development. In certain embodiments, the endogenous immunoglobulinlocus is a heavy chain locus. In certain other embodiments, theendogenous immunoglobulin locus is a light chain locus. Anotherembodiment provides a non-human cell comprising an inactivatedendogenous Ig locus, wherein the endogenous Ig locus comprises adeletion that impairs formation of a functional variable domain andformation of a constant region capable of driving primary B celldevelopment.

One embodiment provides a DNA construct comprising a first flankingsequence, a transgene, and a second flanking sequence, wherein thetransgene comprises a polynucleotide sequence capable of introducing adeletion in an endogenous Ig locus that impairs formation of afunctional variable domain and formation of a constant region capable ofsupporting primary B cell development. Another embodiment provides a kitcomprising the DNA construct. Another embodiment provides a method forinactivating an endogenous immunoglobulin locus comprising impairing theformation of a functional variable domain, and impairing the formationof a constant region capable of driving primary B cell development.

Another embodiment of the invention provides a method of producing anantibody display library comprising providing a non-human animal havinga genome that comprises a chimeric immunoglobulin heavy chain, whereinthe chimeric heavy chain comprises a non-endogenous heavy chain variabledomain and a chimeric heavy chain constant region; recoveringpolynucleotide sequences from the animal, wherein the polynucleotidesequences encode immunoglobulin light chain variable regions andnon-endogenous immunoglobulin heavy chain variable regions; andproducing an antibody display library comprising the heavy chain andlight chain variable regions. One embodiment of the invention providesan antibody display library comprising immunoglobulin heavy chainvariable regions generated by a non-human animal having a genome thatcomprises a chimeric immunoglobulin heavy chain, wherein the chimericheavy chain comprises a non-endogenous heavy chain variable domain and achimeric heavy chain constant region, wherein the variable regions arederived from chimeric antibodies.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 depicts homologous recombination of BAC C5 and BAC P12 in E.coli.

FIG. 2 depicts the removal of the 70 kb repeat between the two copies ofthe pBeloBAC vector using CRE-recombinase.

FIG. 3 depicts the insertion of Tpn-Zeo 15 kb from the junction of thevector.

FIG. 4 depicts homologous recombination of BAC C5P12 and BAC C20 in E.coli.

FIG. 5 depicts the removal of the 44 kb repeat between the two copies ofthe pBeloBAC vector using CRE-recombinase.

DETAILED DESCRIPTION

Overview

The present invention includes chimeric antibodies, non-human animalsthat produce chimeric or humanized antibodies, methods of producing suchnon-human cells and animals, and compositions and kits comprising theantibodies. In specific embodiments of the invention, the non-humananimals are mammals.

Chimeric antibodies, and antigen-binding fragments thereof, describedherein comprise a non-endogenous variable domain and a chimeric heavychain constant region. In particular embodiments, an IgH chain comprisesone or more non-endogenous V, D and J gene segments, a non-endogenousCH1 domain, and endogenous CH2 and CH3 domains. In certain embodiments,an antibody, or antigen-binding fragment thereof, comprising thechimeric IgH chain described herein further comprises an IgL chainhaving an amino acid sequence encoded for by a non-endogenous nucleotidesequence. In other embodiments, an antibody, or antigen-binding fragmentthereof, comprising the chimeric IgH chain described herein furthercomprises an IgL chain having an amino acid sequence encoded for byendogenous and non-endogenous nucleotide sequences.

Engineering the chimeric antibodies in this manner prevents alterationin the V domain conformation resulting from the in vitro switch from afirst C region, particularly a CH1 domain and optionally a portion ofthe hinge region from one species, e.g., mouse, with which it wasevolved during the in vivo immune response to a second C region,particularly a CH1 domain and optionally a portion of the hinge regionfrom another species, e.g., human. The antibodies produced by theanimals of the present invention do not exhibit the reduction or loss ofactivity and potency seen in antibodies from other chimeric antibodyproducing animals when, for example, the human V region is appended to ahuman C region to make a fully human antibody, which may be caused byaltered conformation of the VH domain resulting from the changing of theCH1 domain and/or by differences in antigen binding because of changedlength or flexibility of the upper hinge regions (the peptide sequencefrom the end of the CH1 to the first cysteine residue in the hinge thatforms an inter-heavy chain disulfide bond, and which are variable inlength and composition) when switching from mouse to human constantregion (Roux et al., J. Immunology (1997) 159:3372-3382 and referencestherein). The middle hinge region is bounded by the cysteine residuesthat form inter-heavy chain disulfide bonds.

Definitions

Before describing certain embodiments in detail, it is to be understoodthat this invention is not limited to particular compositions orbiological systems, which can vary. It is also to be understood that theterminology used herein is for the purpose of describing particularillustrative embodiments only, and is not intended to be limiting. Theterms used in this specification generally have their ordinary meaningin the art, within the context of this invention and in the specificcontext where each term is used. Certain terms are discussed below orelsewhere in the specification, to provide additional guidance to thepractitioner in describing the compositions and methods of the inventionand how to make and use them. The scope and meaning of any use of a termwill be apparent from the specific context in which the term is used. Assuch, the definitions set forth herein are intended to provideillustrative guidance in ascertaining particular embodiments of theinvention, without limitation to particular compositions or biologicalsystems. As used in the present disclosure and claims, the singularforms “a,” “an,” and “the” include plural forms unless the contextclearly dictates otherwise.

As used herein, “antibody” and “immunoglobulin” (Ig) are usedinterchangeably herein and refer to protein molecules produced by Bcells that recognize and bind specific antigens and that may either bemembrane bound or secreted. Antibodies may be monoclonal, in that theyare produced by a single clone of B cells and therefore recognize thesame epitope and have the same nucleic acid and amino acid sequence, orpolyclonal, in that they are produced by multiple clones of B cells,recognize one or more epitopes of the same antigen and typically havedifferent nucleic acid and amino acid sequences.

Antibody, or Ig, molecules are typically comprised of two identicalheavy chains and two identical light chains linked together throughdisulfide bonds. There are two types of IgL, Igκ and Igλ. Both heavychains (IgH) and light chains (IgL) contain a variable (V) region ordomain and a constant (C) region or domain. The portion of the IgH locusencoding the V region comprises multiple copies of variable (V),diversity (D), and joining (J) gene segments. The portion of the IgLloci, Igκ and Igλ, encoding the V region comprises multiple copies of Vand J gene segments. The V region encoding portion of the IgH and IgLloci undergo gene rearrangement, e.g., different combinations of genesegments arrange to form the IgH and IgL variable regions, to developdiverse antigen specificity in antibodies. The secreted form of the IgHC region is made up of three C domains, CH1, CH2, CH3, optionally CH4(Cμ), and a hinge region. The membrane-bound form of the IgH C regionalso has membrane and intra-cellular domains. The IgH constant regiondetermines the isotype of the antibody, e.g. IgM, IgD, IgG1, IgG2, IgG3,IgG4, IgA and IgE in humans. It will be appreciated that non-humanmammals encoding multiple Ig isotypes will be able to undergo isotypeclass switching.

A “Fab” domain or fragment comprises the N-terminal portion of the IgH,which includes the V region and the CH1 domain of the IgH, and theentire IgL. A “F(ab′)2” domain comprises the Fab domain and a portion ofthe hinge region, wherein the 2 IgH are linked together via disulfidelinkage in the middle hinge region. Both the Fab and F(ab′)2 are“antigen-binding fragments.” The C-terminal portion of the IgH,comprising the CH2 and CH3 domains, is the “Fc” domain. The Fc domain isthe portion of the Ig recognized by cell receptors, such as the FcR, andto which the complement-activating protein, C1q, binds. The lower hingeregion, which is encoded in the 5′ portion of the CH2 exon, providesflexibility within the antibody for binding to FcR receptors.

As used herein “chimeric antibody” refers to an antibody encoded by apolynucleotide sequence containing polynucleotide sequences derived fromtwo or more species.

A “humanized” antibody is a chimeric antibody that has been engineeredso as to comprise more human sequence than its parental molecule.Humanized antibodies are less immunogenic after administration to humanswhen compared to non-humanized antibodies prepared from another species.For example, a humanized antibody may comprise the variable region of achimeric antibody appended to a human constant region. Chimericantibodies described herein can be used to produce a fully humanantibody.

As used herein “chimeric Ig chain” refers to an Ig heavy chain or an Iglight chain encoded by a polynucleotide sequence containingpolynucleotide sequences derived from two or more species. For example,a chimeric Ig heavy chain may comprise human VH, DH, JH, and CH1 genesegments and mouse CH2 and CH3 gene segments.

“Polypeptide,” “peptide” or “protein” are used interchangeably todescribe a chain of amino acids that are linked together by chemicalbonds. A polypeptide or protein may be an IgH, IgL, V domain, C domain,or an antibody.

“Polynucleotide” refers to a chain of nucleic acids that are linkedtogether by chemical bonds. Polynucleotides include, but are not limitedto, DNA, cDNA, RNA, mRNA, and gene sequences and segments.Polynucleotides may be isolated from a living source such as aeukaryotic cell, prokaryotic cell or virus, or may be derived through invitro manipulation by using standard techniques of molecular biology, orby DNA synthesis, or by a combination of a number of techniques.

“Locus” refers to a location on a chromosome that comprises one or moregenes or exons, such as an IgH or Igκ locus, the cis regulatoryelements, and the binding regions to which trans-acting factors bind. Asused herein, “gene” or “gene segment” refers to the polynucleotidesequence encoding a specific polypeptide or portion thereof, such as aVL domain, a CH1 domain, an upper hinge region, or a portion thereof. Asused herein, “gene segment” and “exon” may be used interchangeably andrefer to a polynucleotide encoding a peptide, or a portion thereof. Agene, or gene segment, may further comprise one or more introns,transcriptional control elements, e.g., promoters, enhancers, or othernon-coding regions (e.g., cis regulatory elements, e.g., 5′ and/or 3′untranslated regions, poly-adenylation sites).

As used herein, the term “inactivated Ig locus” refers to an Ig locusthat does not encode a functional Ig chain. A “functional variableregion” produce from an Ig locus refers to a polynucleotide sequencecapable of undergoing V-(D)-J recombination, being transcribed and saidtranscript being translated into a variable region polypeptide that iscapable of being expressed on a cell surface. A “functional heavy chainconstant region” refers to a constant region capable of beingoperationally joined to a variable region and driving primary B celldevelopment. Primary B cell development refers to the development of Bcells in the primary lymphoid organs, e.g., bone marrow, and encompassesthe transition from stem cell to immature B cell, including thedevelopmental stages of early pro-B cell (i.e., IgH D-J rearranging),late pro-B cell (i.e., IgH V-DJ rearranging), large pre-B cell (i.e.,expresses pre-B receptor), and small pre-B cell (i.e., IgL V-Jrearranging). By “driving” primary B cell development, it is meant thatthe functional heavy chain constant region is capable of, e.g.,anchoring to the cell membrane, signal transduction, and/or binding anFc receptor. A “functional light chain constant region” refers to aconstant region capable of being operationally joined to a variableregion and binding to heavy chain to advance B cell development beyondthe small pre-B cell stage.

“Impair” refers to the introduction of a deletion or mutation thatresults in, e.g., a variable region that is no longer functional or aconstant region that is no longer function. For example, homozygousdeletion of C impairs an IgH from driving primary B cell development insome mammals and strains thereof.

“Mutation” refers to a change in a naturally occurring polynucleotide orpolypeptide sequence. A mutation may result in a functional change.Mutations include both the addition of nucleotides and the deletion ofnucleotides. “Deletion” refers to the removal of one or more nucleotidesfrom the naturally occurring endogenous polynucleotide sequence.Deletions and additions may introduce a frameshift mutation. Deletionsmay also remove entire genes, gene segments or modules. In someinstances, a deletion of part of the naturally occurring endogenoussequence may coincide with the addition of a non-endogenous sequence.For example, a portion of the endogenous polynucleotide sequence may beremoved, i.e., deleted, upon homologous recombination with apolynucleotide comprising a non-endogenous sequence, e.g., a selectionmarker. In other aspects, a deletion of an endogenous polynucleotidesequence may occur after the introduction of two non-endogenousrecognition sequence for a site-specific recombinase, e.g., a loxP site,followed by exposure to the recombinase, e.g., CRE.

The term “endogenous” refers to a polynucleotide sequence which occursnaturally within the cell or animal. “Orthologous” refers to apolynucleotide sequence that encodes the corresponding polypeptide inanother species, e.g., a human CH1 domain and a mouse CH1 domain. Theterm “syngeneic” refers to a polynucleotide sequence that is foundwithin the same species that may be introduced into an animal of thatsame species, e.g., a mouse Vκ gene segment introduced into a mouse. Itshould be noted that the polynucleotide sequence from two individuals ofthe same species but of different strains may have regions ofsignificant difference.

As used herein, the term “homologous” or “homologous sequence” refers toa polynucleotide sequence that has a highly similar sequence, or highpercent identity (e.g. 30%, 40%, 50%, 60%, 70%, 80%, 90% or more), toanother polynucleotide sequence or segment thereof. For example, a DNAconstruct of the invention may comprise a sequence that is homologous toa portion of an endogenous DNA sequence to facilitate recombination atthat specific location. Homologous recombination may take place inprokaryotic and eukaryotic cells.

As used herein, “flanking sequence” or “flanking DNA sequence” refers toa DNA sequence adjacent to a non-endogenous DNA sequence in a DNAconstruct that is homologous to an endogenous DNA sequence or apreviously recombined non-endogenous sequence, or a portion thereof. DNAconstructs of the invention may have one or more flanking sequences,e.g., a flanking sequence on the 3′ and 5′ end of the non-endogenoussequence or a flanking sequence on the 3′ or the 5′ end of thenon-endogenous sequence. The flanking sequence may be homologous to anendogenous sequence within an endogenous gene, or the flanking sequencemay be homologous to an endogenous sequence adjacent to (i.e., outsideof) an endogenous gene.

The phrase “homologous recombination-competent cell” refers to a cellthat is capable of homologously recombining DNA fragments that containregions of overlapping homology. Examples of homologousrecombination-competent cells include, but are not limited to, inducedpluripotent stem cells, hematopoietic stem cells, bacteria, yeast,various cell lines and embryonic stem (ES) cells.

A “non-human animal” refers to any animal other than a human such as,e.g., avians, reptiles and mammals. “Non-human mammal” refers to ananimal other than humans which belongs to the class Mammalia. Examplesof non-human mammals include, but are not limited to, non-humanprimates, camelids, rodents, bovines, ovines, equines, dogs, cats,goats, sheep, dolphins, bats, rabbits, and marsupials. Preferrednon-human mammals rely primarily on somatic hypermutation and/or geneconversion to generate antibody diversity, e.g., mouse, rabbit, pig,sheep, goat, camelids, rodents and cow. Particularly preferred non-humanmammals are mice.

The term “transgenic” refers to a cell or animal comprising anon-endogenous polynucleotide sequence, e.g., a transgene derived fromanother species, incorporated into its genome. For example, a mousewhich contains a human VH gene segment integrated into its genomeoutside the endogenous mouse IgH locus is a transgenic mouse; and amouse which contains a human VH gene segment integrated into its genomedirectly replacing an endogenous mouse VH in the endogenous mouse IgHlocus is a transgenic mouse, sometimes also referred to as a “knock-in”mouse. In transgenic cells and non-human mammals, the non-endogenouspolynucleotide sequence may either be expressed with the endogenousgene, ectopically in the absence of the endogenous gene or in theabsence of the corresponding, or orthologous, endogenous sequenceoriginally found in the cell or non-human mammal.

As used herein, “replace” refers to both direct and functionalreplacement. By “direct replacement” it is meant that an endogenous DNAsequence is replaced with an engineered DNA sequence that comprises anon-endogenous sequence at the location of the endogenous sequence inthe genome, such as by homologous recombination. For example, theendogenous DNA sequence is removed via homologous recombination, or theendogenous sequence remaining between two incorporated non-endogenoussequences is deleted. By “functional replacement” it is meant that thefunction (e.g., as performed by the polypeptide produced from theengineered DNA sequence) of an endogenous DNA sequence is carried out bya non-endogenous DNA sequence. For example, an endogenous IgH locus canbe functionally replaced by a transgene that encodes a chimeric IgHchain and that is inserted into the genome outside of the endogenous IgHlocus.

A “humanized” animal, as used herein refers to a non-human animal, e.g.,a mouse, that has a composite genetic structure that retains genesequences of the non-human animal, in addition to one or more genesegments and or gene regulatory sequences of the original genetic makeuphaving been replaced with analogous human sequences.

As used herein, the term “vector” refers to a nucleic acid molecule intowhich another nucleic acid fragment can be integrated without loss ofthe vector's ability to replicate. Vectors may originate from a virus, aplasmid or the cell of a higher organism. Vectors are utilized tointroduce foreign or recombinant DNA into a host cell, wherein thevector is replicated.

A polynucleotide agent can be contained in a vector, which canfacilitate manipulation of the polynucleotide, including introduction ofthe polynucleotide into a target cell. The vector can be a cloningvector, which is useful for maintaining the polynucleotide, or can be anexpression vector, which contains, in addition to the polynucleotide,regulatory elements useful for expressing the polynucleotide and, wherethe polynucleotide encodes an RNA, for expressing the encoded RNA in aparticular cell, either for subsequent translation of the RNA into apolypeptide or for subsequent trans regulatory activity by the RNA inthe cell. An expression vector can contain the expression elementsnecessary to achieve, for example, sustained transcription of theencoding polynucleotide, or the regulatory elements can be operativelylinked to the polynucleotide prior to its being cloned into the vector.

An expression vector (or the polynucleotide) generally contains orencodes a promoter sequence, which can provide constitutive or, ifdesired, inducible or tissue specific or developmental stage specificexpression of the encoding polynucleotide, a poly-A recognitionsequence, and a ribosome recognition site or internal ribosome entrysite, or other regulatory elements such as an enhancer, which can betissue specific. The vector also can contain elements required forreplication in a prokaryotic or eukaryotic host system or both, asdesired. Such vectors, which include plasmid vectors and viral vectorssuch as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus,vaccinia virus, alpha virus and adeno-associated virus vectors, are wellknown and can be purchased from a commercial source (Promega, MadisonWis.; Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.) or canbe constructed by one skilled in the art (see, for example, Meth.Enzymol., Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly,Canc. Gene Ther. 1:51-64, 1994; Flotte, J. Bioenerg. Biomemb 25:37-42,1993; Kirshenbaum et al., J. Clin. Invest 92:381-387, 1993; each ofwhich is incorporated herein by reference).

A DNA vector utilized in the methods of the invention can containpositive and negative selection markers. Positive and negative markerscan be genes that when expressed confer drug resistance to cellsexpressing these genes. Suitable selection markers for E. coli caninclude, but are not limited to: Km (Kanamycin resistant gene), tetA(tetracycline resistant gene) and beta-lactamase (ampicillin resistantgene). Suitable selection markers for mammalian cells in culture caninclude, but are not limited to: hyg (hygromycin resistance gene), puro(puromycin resistance gene) and G418 (neomycin resistance gene). Theselection markers also can be metabolic genes that can convert asubstance into a toxic substance. For example, the gene thymidine kinasewhen expressed converts the drug gancyclovir into a toxic product. Thus,treatment of cells with gancylcovir can negatively select for genes thatdo not express thymidine kinase.

In a related aspect, the selection markers can be “screenable markers,”such as green fluorescent protein (GFP), yellow fluorescent protein(YFP), red fluorescent protein (RFP), GFP-like proteins, and luciferase.

Various types of vectors are available in the art and include, but arenot limited to, bacterial, viral, and yeast vectors. A DNA vector can beany suitable DNA vector, including a plasmid, cosmid, bacteriophage,p1-derived artificial chromosome (PAC), bacterial artificial chromosome(BAC), yeast artificial chromosome (YAC), or mammalian artificialchromosome (MAC). In certain embodiments, the DNA vector is a BAC. Thevarious DNA vectors are selected as appropriate for the size of DNAinserted in the construct. In one embodiment, the DNA constructs arebacterial artificial chromosomes or fragments thereof.

The term “bacterial artificial chromosome” or “BAC” as used hereinrefers to a bacterial DNA vector. BACs, such as those derived from E.coli, may be utilized for introducing, deleting or replacing DNAsequences of non-human mammalian cells or animals via homologousrecombination. E. coli can maintain complex genomic DNA as large as 500kb or greater in the form of BACs (see Shizuya and Kouros-Mehr, Keio JMed. 2001, 50(1):26-30), with greater DNA stability than cosmids oryeast artificial chromosomes. In addition, BAC libraries of human DNAgenomic DNA have more complete and accurate representation of the humangenome than libraries in cosmids or yeast artificial chromosomes. BACsare described in further detail in U.S. application Ser. Nos. 10/659,034and 61/012,701, which are hereby incorporated by reference in theirentireties.

DNA fragments comprising an Ig locus, or a portion thereof, to beincorporated into the non-human mammal are isolated from the samespecies of non-human mammal prior to humanization of the locus. MultipleBACs containing overlapping fragments of an Ig locus can be humanizedand the overlapping fragments recombine to generate a continuous IgH orIgL locus. The resulting chimeric Ig locus comprises the human genesegments operably linked to the non-human mammal Ig gene segments toproduce a functional Ig locus, wherein the locus is capable ofundergoing gene rearrangement and thereby producing a diversifiedrepertoire of chimeric antibodies.

These processes for recombining BACs and/or of engineering a chimeric Iglocus or fragment thereof requires that a bacterial cell, such as E.coli, be transformed with a BAC containing the host Ig locus or aportion thereof. The BAC containing bacillus is then transformed with arecombination vector comprising the desired human Ig gene segment linkedto flanking homology sequence shared with the BAC containing the host Iglocus or portion thereof. The shared sequence homology mediateshomologous recombination and cross-over between the human Ig genesegment on the recombination vector and the non-human mammal Ig genesegment on the BAC. Detection of homologously recombined BACs mayutilize selectable and/or screenable markers incorporated into thevector. Humanized BACs can be readily isolated from the bacteria andused for producing knock-in non-human cells. Methods of recombining BACsand engineering insertions and deletions within DNA on BACs and methodsfor producing genetically modified mice therefrom are documented. See,e.g., U.S. Pat. No. 5,770,429; Fishwild, D. et al. (1996) Nat.Biotechnol. 14:845-851; Valenzuela et al. Nature Biotech. (2003)21:652-659; Testa et al. Nature Biotech. (2003) 21:443-447; and Yang andSeed. Nature Biotech. (2003) 21:447-451.

The first recombination step may be carried out in a strain of E. colithat is deficient for sbcB, sbcC, recB, recC or recD activity and has atemperature sensitive mutation in recA. After the recombination step, arecombined DNA construct is isolated, the construct having the varioussequences and orientations as described.

The regions used for BAC recombineering should be a length that allowsfor homologous recombination. For example, the flanking regions may befrom about 0.1 to 19 kb, and typically from about 1 kb to 15 kb, orabout 2 kb to 10 kb.

The process for recombining BACs to make larger and/or tailored BACscomprising portions of the Ig loci requires that a bacterial cell, suchas E. coli, be transformed with a BAC carrying a first Ig locus, aportion thereof, or some other target sequence. The BAC containing E.coli is then transformed with a recombination vector (e.g., plasmid orBAC) comprising the desired Ig gene segment to be introduced into thetarget DNA, e.g., one or more human VH, DH and/or JH gene segments to bejoined to a region from the mouse IgH locus, both of which vectors havea region of sequence identity. This shared region of identity in thepresence of functional recA in the E. coli mediates cross-over betweenthe Ig gene segment on the recombination vector and the non-human mammalIg gene segment on the BAC. Selection and resolution of homologouslyrecombined BACs may utilize selectable and/or screenable markersincorporated into the vectors. Humanized and chimeric BACs can bereadily purified from the E. coli and used for producing transgenic andknock-in non-human cells and animals by introducing the DNA by variousmethods known in the art and selecting and/or screening for eitherrandom or targeted integration events.

Alternatively, the DNA fragments containing an Ig locus to beincorporated into a non-human animal are derived from DNA synthesized invitro. The genomes of many organisms have been completely sequenced(e.g., human, chimpanzee, rhesus monkey, mouse, rat, dog, cat, chicken,guinea pig, rabbit, horse, cow, alpaca) and are publicly available withannotation. For many other organisms, there is publicly availableinformation on the sequences of the transcriptome. In particular but notlimited to, the human and mouse immunoglobulin loci have been studiedand characterized for the location and activity of coding gene segmentsand non-coding regulatory elements.

The term “in silico,” as used herein, refers to the use of a computer orcomputer algorithm to model a naturally occurring or in vitro process,and in particular, to assist in the design of a nucleotide orpolypeptide sequence and/or the synthetic production of a nucleotide orpolypeptide sequence using, all or in part, a cell free system (e.g.,using automated chemical synthesis). The sequences of the Ig loci may bemanipulated and recombined in silico using commonly available softwarefor nucleic acid sequence analysis. In silico recombination may bewithin the same locus, between two loci from the same species, orbetween loci from two or more species. In silico recombination may beperformed to design either a functional sequence or a non-functional,inactivated sequence. Precise nucleotide-by-nucleotide engineeringallows for precise manipulation of sequence composition that can beapplied to precisely engineer the function of the transgene and aftertranscription and translation, result in precisely engineeredcomposition and function of the polypeptide product of the locus.

Sequences of an Ig locus may also be recombined in silico with thosefrom a non-immunoglobulin locus, either from the same or a differentspecies. Such sequences include, but are not limited to, genes forpositive and negative drug selection markers such as G418, hyg, puro andtk, site-specific recombinase recognition sequences such lox P sites andits variants and frt sites, and precisely demarcated sequences fordriving homologous recombination. After assembling the desired sequencein silico, it may then be synthesized and assembled without errors(Kodumal et al., Proc. Natl. Acad. Sci. (2004) 101:15573-15578). Thesynthesis, assembly and sequencing of large DNAs are provided on acontractual basis (e.g., DNA 2.0, Menlo Park, Calif.; Blue HeronBiotechnology, Bothell, Wash.; and Eurogentec, San Diego, Calif.). Suchsynthetic DNA sequences are carried in vectors such as plasmids and BACsand can be transferred into other vectors such as YACs.

The term “construct” as used herein refers to a sequence of DNAartificially constructed by genetic engineering, recombineering orsynthesis. Constructs include, for example, transgenes and vectors(e.g., BACs, P1s, lambda bacteriophage, cosmids, plasmids, YACs andMACs). In one embodiment, the DNA constructs are linearized prior tointroduction into a cell. In another embodiment, the DNA constructs arenot linearized prior to introduction into a cell.

As used herein, “loxP” and “CRE” refer to a site-specific recombinationsystem derived from P1 bacteriophage. loxP sites are 34 nucleotides inlength. When DNA is flanked on either side by a loxP site and exposed toCRE mediated recombination, the intervening DNA is deleted and the twoloxP sites resolve to one. The use of the CRE/lox system, includingvariant-sequence lox sites and variants of CRE, for which geneticengineering in many species, including mice, is well documented.

A similar system, employing frt sites and flp recombinase from S.cerevisiae can be employed to similar effect. As used herein, anyimplementation of CRE/loxP to mediate deletional events in mammaliancells in culture can also be mediated by the flp/frt system.

As used herein the terms “immunize,” “immunization,” and “immunizing”refer to exposing the adaptive immune system of an animal to an antigen.The antigen can be introduced using various routes of administration,such as injection, inhalation, ingestion or DNA immunization. Upon asecond exposure to the same antigen, the adaptive immune response, i.e.T cell and B cell responses, is enhanced.

“Antigen” refers to a peptide, lipid, amino acid, nucleic acid,saccharide, hapten or chemical entity that is recognized by the adaptiveimmune system. Examples of antigens include, but are not limited to,bacterial cell wall components, pollen, and rh factor. “Target antigen”refers to an antigen, peptide, lipid, saccharide, or amino acid, whichis recognized by the adaptive immune system that is chosen to produce animmune response against, e.g., a specific infectious agent or endogenousor exogenous cell or product thereof. Target antigens include, but arenot limited to, bacterial and viral components, tumor-specific antigens,cytokines, cell surface molecules, any and all antigens against whichantibodies or other binding proteins have been made by in vivo or invitro methods, etc.

The term “pharmaceutical” or “pharmaceutical drug,” as used hereinrefers to any pharmacological, therapeutic or active biological agentthat may be administered to a subject or patient. In certain embodimentsthe subject is an animal, and preferably a mammal, most preferably ahuman.

The term “pharmaceutically acceptable carrier” refers generally to anymaterial that may accompany the pharmaceutical drug and which does notcause an adverse reaction with the subject's immune system.

The term “administering,” as used herein, refers to any mode oftransferring, delivering, introducing, or transporting a pharmaceuticaldrug or other agent, such as a target antigen, to a subject. Such modesinclude oral administration, topical contact, intravenous,intraperitoneal, intramuscular, intranasal, or subcutaneousadministration.

Non-Human Mammals and Cells Encoding Chimeric IG Heavy Chains

Non-human animals and cells of the present invention comprise one ormore altered Ig loci (e.g., IgH, Igκ, and/or Igλ) comprisingnon-endogenous Ig gene segments that replace the endogenous genesegments. In certain embodiments, the altered loci directly replace theendogenous gene segments. In other embodiments, the altered locifunctionally replace the endogenous gene segments.

The non-endogenous gene segments may be derived from any species, andmay include syngeneic gene segments. The non-endogenous sequence may bederived from, for example, humans, mice, non-human primates, camelids,rodents, bovines, ovines, equines, dogs, cats, goats, sheep, dolphins,bats, rabbits, and marsupials. As described above, the non-human cell oranimal may be any non-human animal. Accordingly, the transgenic cellsand animals described herein may comprise DNA sequences derived from anycombination of species, provided that the animal is a non-human mammal.By way of example, chimeric mouse cells and mice comprising human orcamelid Ig polynucleotide sequences are envisioned. In addition, thetransgenic cell or animal may comprise non-endogenous DNA from more thanone species. For example, a transgenic mouse genome can comprise bothhuman and camelid DNA sequences.

The transgenic cells and animals described herein comprise one or morenon-endogenous V gene segments. In specific embodiments, the preferrednon-human animal is a mammal. In certain embodiments, the cell or animalfurther comprises one or more non-endogenous J gene segments. In anotherembodiment, a cell or animal comprising a chimeric IgH chain optionallyfurther comprises one or more non-endogenous D gene segments.

In one embodiment, the cell or animal comprises a genome encoding achimeric IgH chain and a transgenic light chain. The transgenic lightchain may be an Igκ or an Igλ light chain. In addition, the transgeniclight chain may be chimeric, or the transgenic light chain may compriseonly non-endogenous amino acid sequences. In particular embodiments, thecell or animal comprises a genome encoding non-endogenous IgH, Igκ andIgλ gene segments. The transgenic cells and mammals comprising achimeric IgH chain described herein comprise a non-endogenous CH1 domainthat replaces a CH1 domain in a specific endogenous CH gene, e.g., Cμ,Cδ, or Cγ. In certain embodiments, the non-endogenous CH1 domain isorthologous to the endogenous CH region. In other embodiments, thenon-endogenous CH1-domain is not orthologous to the endogenous CHregion. In another embodiment, more than one endogenous CH1 domain isreplaced with a non-endogenous CH1 domain. In a related embodiment, allof the endogenous CH1 domains are replaced with a non-endogenous CH1domain. For example, an orthologous human CH1 may replace each of theendogenous Cγ genes (e.g., human Cγ1 CH1 replaces mouse Cγ1 CH1 andhuman Cγ2 CH1 replaces mouse Cγ2 CH1 etc.). In another embodiment, theCH1 domain that replaces the CH1 domain of each of the endogenous Cγgenes is a single human IgG isotype more frequently used in therapeuticmAbs, typically Cγ1, Cγ2 or Cγ4, so as to better facilitate in vivomaturation of a human V domain in the context of a more clinicallyrelevant human CH1 domain.

Optionally, the upper hinge sequences of the endogenous C genes may alsobe replaced with orthologous non-endogenous C hinge sequences.Alternatively, the upper and middle hinge sequences of the endogenous Cgenes may also be replaced with the orthologous non-endogenous C hingesequences, respectively. If human middle hinge regions are used, thehuman Cγ4 middle hinge sequence may be engineered to contain a prolineat residue at position 229 rather than a serine in order to driveinter-heavy chain dimerization via disulfide bonds. The lower hingeregion, a part of the CH2 domain, of the endogenous Cγ gene is notreplaced in order to facilitate optimal binding to an endogenous FcγR.These three optional engineering strategies provide a non-endogenousheavy chain Fab domain, Fab domain plus upper hinge, or F(ab′)2,respectively. If the upper are replaced with human upper hinge regions,the variable region of the resulting antibody is more likely to retainoptimal characteristics upon conversion to fully human IgG.

Another embodiment incorporates fully non-endogenous, e.g., human, Igincluding the C regions comprising CH1-hinge-CH2-CH3(-CH4) and thecognate syngeneic, e.g., mouse, membrane and intracellular domains so asto provide native intracellular signal transduction and to enableassociation of the IgH in the B-cell receptor with Igα and Igβ andtherein allow endogenous-type signaling from the Igα, Igβ and IgGcontaining B-cell receptor. In yet another embodiment, the membrane andintracellular domain of the heavy chain constant region are from thesame or non-cognate syngeneic heavy chain isotypes. Such engineering ofthe constant region genes can be readily accomplished using methods ofthe invention as detailed below.

In yet another embodiment, the transgenic cells and animals comprising achimeric IgH chain described herein comprise constant region encoded bya non-endogenous polynucleotide sequence derived from two or morespecies. For example, a transgenic mouse having a genome encoding achimeric IgH chain constant region comprises a human CH1 domain, humanupper hinge regions, and rat CH2 and CH3 domains, is envisioned. Inanimals having a xenogeneic constant region, it is preferred that theconstant region is capable of interacting with (e.g., binding) anendogenous FcR.

In yet another embodiment, the transgenic cells and animals comprising achimeric IgH chain described herein comprise constant region encoded bya non-endogenous polynucleotide sequence and endogenous polynucleotidesequence derived from two strains. For example, a transgenic mousehaving a genome encoding a chimeric IgH chain constant region comprisesa human CH1 domain, human upper hinge regions, and Balb/c mouse CH2 andCH3 coding sequences embedded into C57BL/6 (“B6”) genomic DNA,comprising all B6 genetic information except that Balb/c-sequence exonsfor CH2 and CH3 replace their B6 counterparts, is envisioned.

In one embodiment, the composite IgH sequence comprises at least 3 kbupstream of the VH6 promoter through the D gene cluster through 3′ ofJH6 and is all human and in germline configuration. In anotherembodiment, the composite IgH sequence comprises at least 3 kb upstreamof the VH6 promoter through the D gene cluster through 3′ of JH6 and isall human and in germline configuration except that the D gene clusteris replaced by all or part of that of a xenogeneic species. In anotheraspect of the invention, there are additional human VH genes upstream ofhuman VH6. In yet another aspect, the additional VH genes are ingermline configuration. In an alternative aspect, the additional VHgenes are sizes less than that in the human genome, unit sizes thatcomprise upstream regulatory elements such as cis-regulatory elementsand binding sites for trans-acting factors, coding sequences, intronsand 500 bp downstream of the last codon of each VH. In one aspect, theunit size is 10 kb or less. In another aspect, the unit size is 5 kb orless. In another aspect, the VH genes are selected from the subset ofcommonly shared VH genes amongst human haplotypes. In another aspect, VHgenes, DH genes and JH genes are chosen to reflect a specific allelesuch as the most prevalent allele in human populations. In yet anotheraspect, the individual codons of the VH gene are codon-optimized forefficient expression in a specific non-human mammal. In another aspectthe individual codons are optimized to be a template for somatichypermutation.

In another embodiment, the composite IgH sequence comprises mouse DNAsequence starting at least 3 kb upstream of the promoter for thefunctional VH gene nearest the D gene cluster, e.g., VH5-2, through 3′of JH4 in germline configuration and into which the coding sequenceshave been replaced, all or in part, by human coding sequences, e.g.,coding sequence for mouse VH5-2 is replaced by coding sequence for humanVH6-1, mouse DH coding sequences replaced by human DH coding sequencesand mouse JH coding sequences replaced by human JH coding sequences. Ininstances in which the number of human coding elements exceeds those inthe mouse, e.g., 6 human JH coding sequences versus 4 mouse JH codingsequences, the additional JH genes may be included by various means,e.g., inserting the additional human JH coding sequences with their cisregulatory elements, such as recombination signal sequences downstreamof the JH4, or omitted altogether.

In other embodiments, the mouse VH coding sequences are replaced, all orin part, by human VL coding sequences. In some embodiments, the entireDH gene cluster is of mouse sequence. In other embodiments, the entireDH gene cluster is of xenogeneic species. In another aspect of theinvention, there are additional VH genes upstream of VH6 codingsequences, such that the all of the sequence is mouse except that codingsequences of functional VH genes are replaced with that of human VHgenes.

In yet another aspect, the additional VH genes are in germlineconfiguration. In an alternative aspect, the additional VH genes aresizes less than that in the mouse genome, unit sizes that compriseupstream regulatory elements such as cis-regulatory elements and bindingsites for trans-acting factors, coding sequences, introns and 500 bpdownstream of the last codon of each VH. In one aspect, the unit size is10 kb or less. In another aspect, the unit size is 5 kb or less.

In another aspect, the VH genes are selected from a subset known to befunctional, with the replacing human VH gene coding sequence being froma known functional human VH gene and replacing the mouse VH gene codingsequence of a known functional mouse VH gene. In another aspect, thehuman VH coding sequences are selected from the subset of commonlyshared VH genes amongst human haplotypes. In another aspect, thereplacing VH coding sequences, DH coding sequences and JH codingsequences are chosen to reflect a specific allele such as the mostprevalent allele in human populations. In another aspect, some or all ofthe replacing VH coding sequences, DH coding sequences and JH codingsequences are from a xenogeneic species other than human. In yet anotheraspect, the individual codons of the VH gene are codon-optimized forefficient expression in a specific non-human mammal. In another aspectthe individual codons are optimized to be a template for somatichypermutation.

In another embodiment, the composite IgH sequence further comprises 3′of the most 3′ JH the mouse sequence immediately downstream of mouse JH4through Eμ through Cμ through Cδ through immediately 5′ of the mouse Cγ3promoter all in germline configuration with the exception of thereplacement of the CH1 domains of mouse Cμ and Cδ by their humancounterparts. In some instances, the mouse upper hinge regions arereplaced by their respective human upper hinge regions. In a furtherembodiment, the mouse Cγ genes are configured in germline configurationwith the exception of the replacement of their CH1 domains by human CH1domains.

In some instances, the mouse upper hinge regions are replaced by humanupper hinge regions. In some embodiments, the mouse Cγ3 coding sequencesare replaced by human CH1 and mouse CH2, CH3, membrane and intracellulardomains from Cγ1. In another embodiment, the completegermline-configured mouse Cγ3 sequence from the promoter upstream of theswitch region through the intracellular domains and 3′ untranslatedsequence and poly(A) site are replaced by the complete correspondingsequences from Cγ1 in germline configuration with human CH1 replacingmouse CH1 from Cγ1 to effectively replace the complete Cγ3 gene bychimeric Cγ1. In some embodiments, a mouse constant coding sequence isreplaced by human CH1 and mouse CH2, CH3, membrane and intracellulardomains from different mouse constant region isotypes, e.g., CH2, CH3and membrane domains from mouse Cγ2a and intracellular domain from Cμ.In still other embodiments the sequence of the CH2 and CH3 domains arefurthered modified to modulate binding to Fc receptors, such asdiminished binding to the inhibitory receptor, FcγR2b, therein producinga stronger secondary immune response.

In another embodiment, the cell or non-human animal comprises a locusencoding a human Ig light chain comprising a human Igκ variable region.In a related embodiment, the Ig light chain locus further comprises ahuman Igκ constant region. In one embodiment, the composite Igκ sequencecomprises mouse DNA sequence from at least 3 kb upstream of the promoterof the Vκ gene most proximal to mouse Jκ1 (Vκ3-1) through 3′ of mouseJκ5 and is in germline configuration and into which the coding sequenceshave been replaced, all or in part, by human coding sequences, e.g.,coding sequence for mouse Vκ3-1 is replaced by coding sequence for humanVκ4-1 and mouse Jκ coding sequences replaced by human Jκ codingsequences. In another embodiment the sequence from Jκ5 through Cκ ismouse and in germline configuration and into which the Cκ codingsequences have been replaced, all or in part, by human coding sequences.

In another aspect there is a 3′LCR region and RS element downstream ofthe Cκ gene. In one aspect, the 3′ LCR and RS elements are mouse and ingermline configuration. In another aspect of the invention, there areadditional Vκ genes upstream of the coding sequences for human Vκ4-1,such that all of the sequence is mouse except that coding sequences offunctional Vκ genes are replaced with that of human Vκ genes.

In yet another aspect, the additional Vκ genes are in germlineconfiguration. In an alternative aspect, the additional Vκ genes aresizes less than that in the mouse genome, unit sizes that compriseupstream regulatory elements such as cis-regulatory elements and bindingsites for trans-acting factors, coding sequences, introns and 500 bpdownstream of the last codon of each Vκ. In one aspect, the unit size is10 kb or less. In another aspect, the unit size is 5 kb or less. Inanother aspect, the Vκ genes are selected from the subset of commonlyshared Vκ genes amongst human haplotypes. In another aspect, Vκ genesand Jκ genes are chosen to reflect a specific allele such as the mostprevalent allele in human populations. In yet another aspect, theindividual codons of the Vκ gene are codon-optimized for efficientexpression in a specific non-human mammal. In another aspect theindividual codons are optimized to be a template for somatichypermutation.

In yet another embodiment, the human Ig light chain locus comprises allor a portion of a human Igλ light chain locus and an Igλ 3′LCR, or afunctional fragment thereof. In one embodiment, the human Igλ lightchain locus comprises the entire human Igλ locus. In another embodimentthe human Igλ light chain locus comprises human Vλ coding sequences and1 to 7 Jλ-Cλ coding sequence pairs, wherein the human Cλ is replacedwith syngeneic Cλ. In yet another embodiment, the human Igλ light chainlocus comprises human Vλ coding sequences, 1 to 7 human Jλ codingsequences, and a single human Cλ coding sequence, wherein the humancoding sequences resemble a human Igλ locus configuration.

In particular embodiments, the Igλ 3′ LCR, or a functional fragmentthereof, is from a mammal selected from the group consisting of human,non-human primate, and rat. In one embodiment the Igλ 3′ LCR, or afunctional fragment thereof, is human. In particular embodiments, theIgλ 3′ LCR, or a functional fragment thereof, binds NFκb. In oneembodiment, the Igλ 3′ LCR, or a functional fragment thereof, is frommouse and has been mutagenized so as to restore binding of NFκb. Inother embodiments, the 3′ LCR, or a functional fragment thereof, in thehuman Igλ locus is an Igκ 3′ LCR, or functional fragment thereof.

In one embodiment, the composite Igλ sequence comprises at least 3 kbupstream of the Vλ 3r promoter through 3′ of Jλ7-Cλ7 and is all humanand in germline configuration. In another aspect, the sequence fromJλ7-Cλ7 through the λ 3′ LCR is human and in germline configuration. Inanother aspect of the invention, there are additional human Vλ genesupstream of human Vλ 3r. In yet another aspect, the additional Vλ genesare in germline configuration. In an alternative aspect, the additionalVλ genes are sizes less than that in the human genome, unit sizes thatcomprise upstream regulatory elements such as cis-regulatory elementsand binding sites for trans-acting factors, coding sequences, intronsand 500 bp downstream of the last codon of each Vλ. In one aspect, theunit size is 10 kb or less. In another aspect, the unit size is 5 kb orless. In another aspect, the Vλ genes are selected from the subset ofcommonly shared Vλ genes amongst human haplotypes. In another aspect, Vλgenes and Jλ genes are chosen to reflect a specific allele such as themost prevalent allele in human populations. In yet another aspect, theindividual codons of the Vλ gene are codon-optimized for efficientexpression in a specific non-human mammal. In another aspect theindividual codons are optimized to be a template for somatichypermutation.

Production of Chimeric Cells and Animals

Specific embodiments of the invention provide methods of producing theanimals and cells. In antibody producing mammals, for example, theendogenous Ig V, (D) and J genes are replaced by non-endogenous (e.g.,human) Ig gene segments. In certain embodiments, the endogenousimmunoglobulin (Ig) V, (D) and J genes are directly replaced bynon-endogenous orthologs. In other embodiments, the endogenous genes arefunctionally replaced by non-endogenous orthologs while the endogenousgenes are inactivated using various techniques as described herein andknown in the art.

For example, one or more constructs carrying large portions of thenon-endogenous V, D and J genes can replace all or a portion of theendogenous V, D and J genes. In certain embodiments, this can be done byhomologously recombining the constructs into or adjacent to each Iglocus. Accordingly, the constructs can replace the endogenous sequencesby sequential (“walking”) replacement or by introducing two constructsinto or adjacent to the endogenous Ig locus and subsequently removingintervening sequences.

An exemplary method of producing a cell having a genome that comprises achimeric immunoglobulin heavy chain, wherein the heavy chain comprises anon-endogenous variable domain and a chimeric constant region, comprisesthe steps of (1) producing a first DNA construct, wherein the firstconstruct comprises one or more non-endogenous VH, DH and/or JH genesegments, a first and a second flanking region, wherein the firstflanking region is homologous to a DNA sequence 5′ of the endogenousimmunoglobulin heavy chain locus, and a first site specificrecombination recognition sequence near the 3′ end of the firstconstruct; (2) producing a second DNA construct, wherein the secondconstruct comprises one or more non-endogenous constant region genesegments, a third and a fourth flanking region, wherein the fourthflanking region is homologous to a DNA sequence 3′ of the endogenousimmunoglobulin heavy chain locus, and a second site specificrecombination recognition sequence near the 5′ end of the secondconstruct; (3) homologously recombining the first and second constructsinto the genome of a cell; and (4) introducing a site-specificrecombinase into the cell, thereby removing an intervening sequencebetween the first and second site-specific recombinase recognitionsequences.

Alternatively, the constructs can be introduced into non-human animalcells by transfection into cells in tissue culture or by pro-nuclearmicroinjection into fertilized eggs, and the non-endogenous sequencesare randomly integrated into the genome. A separate functionalinactivation (i.e., “knock-out”) of the endogenous locus can beperformed by gene targeting in mammalian cells in culture using themethods known in the art or described herein or by other methods such asthe use of engineered zinc-finger nucleases or meganucleases.

A construct carrying all or part of the IgH locus downstream of JH canbe engineered so that in each constant region gene, the endogenous CH1domain is replaced with a non-endogenous CH1 domain. This can beaccomplished by techniques known in the art, such as recombination ofBACs in E. coli or YACs in S. cerevisiae. Such replacement can also beaccomplished using sequential homologous recombination driven knock-inreplacement of the endogenous CH1 domain by the non-endogenous CH1domain. Selectable markers used for the selecting recombinants can beflanked by site-specific recombinase recognition sequences, e.g. loxPsites and deleted via subsequent exposure to the site-specificrecombines, e.g. CRE. Using different variant loxP sites to flank theselectable marker at each step restricts the CRE-mediated deletion toonly the sequence between the specific loxP site and preventslonger-range deletion to an already existing loxP site. Alternatively, aconstruct carrying all or part of the IgH locus downstream of JH can beengineered so that in each constant region gene, the endogenous CH1domain is replaced with a non-endogenous CH1 domain, using the abilityto precisely synthesize and assemble DNAs based on published genomesequences of organisms such as humans and mice. Such synthesis andassembly is known in the art and is practiced by commercial entities(e.g., DNA2.0, Menlo Park, Calif.; Blue Heron Biotechnology, Bothell,Wash.).

According to one method of producing a cell comprising a chimeric heavychain as described herein, a construct comprising the endogenous IgHloci downstream of the J gene cluster, wherein each retained C genecomprises a non-endogenous CH1-endogenous CH2-CH3 (and CH4 for Cμ), andmembrane and intracellular domain exons is generated and introduced intothe genome of a non-human cell. In certain embodiments, the construct ishomologously recombined into or adjacent to the endogenous IgH locus. Inother embodiments, the construct is randomly integrated into the genomeof the cell. The construct may further comprise one or morenon-endogenous V gene segments. In an alternative embodiment, theconstruct comprising the constant region gene segments is introducedinto the genome of the cell either as a first introduction step to befollowed by replacement of the endogenous V-D-J genes withnon-endogenous V gene segments or in the opposite order, i.e.,introduction of non-endogenous V gene segments followed by theintroduction of the construct comprising the constant region genesegments engineered as described herein.

When using more than one construct to introduce non-endogenous Ig genesegments, the content of the Ig locus is not restricted to only constantregion gene segments on one construct and variable region gene segmentson the other. For example, a construct comprising C gene segments mayalso comprise one or more J gene segments, D gene segments and/or V genesegments. Similarly, a construct comprising V gene segments may furthercomprise one or more of D gene segments, J gene segments and/or C genesegments.

Constructs carrying the constant region genes may be engineered invitro, in E. coli or S. cerevisiae or synthesized in vitro prior tointroduction into ES cells so as to delete any unwanted or unneeded genesegments, such as the Cε and Cα genes. This would constrain the animalsto making Cμ and Cδ for primary immune responses and Cγ isotypes forsecondary, affinity-matured immune responses, from which therapeuticantibody candidates would typically be recovered.

In addition, constructs include both coding and non-codingpolynucleotide sequences of which the non-coding polynucleotidesequences may be either non-endogenous or syngeneic polynucleotidesequences. For example, the endogenous (i.e., syngeneic) IgH 3′ locuscontrol region (LCR), or a portion thereof, are included downstream ofthe most 3′ CH gene, Eμ, or a portion thereof is included between themost 3′ JH and Cμ, and all or a portion of the Sμ and Sγ regions,promoters upstream of gene segments such as V gene segments and CHswitch regions and recombination signal sequences (RSS). In addition, itis advantageous to include other intergenic regions that have beenhypothesized to have gene regulation function such as the intergenicregion between the most 3′ VH gene the start of the D cluster and theintergenic region between Cδ and the first Cg switch region.Corresponding elements exist in the Ig light chain loci with documentedfunction and location, e.g., Eκ, Igκ 3′ LCR and Ed. Because theendogenous mouse Igλ light chain locus possesses defective 3′ LCRs, itis advantageous to use an orthologous functional Igλ 3′ LCR from anotherspecies, e.g., human, rodent other than mouse, or to mutate the mouse 3′LCR to restore NFκb binding.

Similar strategies are employed for the endogenous Igκ locus except thata complete non-endogenous Cκ gene can be incorporated in the construct,thus producing fully non-endogenous Igκ chains. A non-endogenous Cλlocus could also be incorporated in a similar manner. For example, aconstruct comprising human Vκ and Cκ gene segments can be generated thatencodes a fully human Igκ chain. Similarly, a construct comprising humanVλ and Cλ gene segments can be generated that encodes a fully human Igλchain.

Yet another aspect of the invention comprises incorporating fully humanIg loci, including human C regions, in place of the complete endogenousIg loci. In an additional embodiment, a cluster of endogenous FcR genesis also replaced with an orthologous cluster of human FcR genes usingsimilar BAC-based genetic engineering in homologous recombinationcompetent cells, such as mouse ES cells. The cluster of endogenous FcγRgenes can be directly replaced in the same ES cell in which the humanIgH locus or portions thereof have replaced the endogenous locus or in aseparate ES cell. Alternatively, the cluster of endogenous FcγR genescan be functionally replaced in the same ES cell in which the human IgHlocus or portions thereof have replaced the endogenous locus or in aseparate ES cell. In the latter instance, mice would be derived fromsaid ES cells and bred with mice carrying the engineered Ig locus (loci)so as to produce mice that make human IgG antibodies that bind to humanFcγR in place of mouse FcγR genes. In either way fully human antibodieswould be produced and during an immune response would be able to engagethe human FcR receptors normally. Such transgenic animals would alsohave the benefit of being useful for testing for the activity andeffector function of human therapeutic mAb candidates in models ofdisease when bred onto the appropriate genetic background for the model,i.e., SCID, nu/nu, nod, and lpr mice. Further, the human target genesequence can replace the endogenous gene using BAC targeting technologyin homologous recombination-competent cells, providing models for targetvalidation and functional testing of the antibody. In this instance, thehuman CH genes may be engineered to have cytoplasmic and/or membranedomain gene segments from mouse or other orthologous species tofacilitate native signal transduction in the B cell. Alternatively toreplacing the entire endogenous FcγR locus with the complete complementof wild-type genes in the human FcγR locus, certain FcγR could bemutated to have attenuated function or deleted entirely. For example,mutation to render the inhibitory human FcγR2b inactive and havingsimultaneous inactivation of the mouse orthologue would render thegenetically engineered mouse carrying both mutations more susceptible todeveloping autoreactive B cells, with a consequent potential benefit ofbroadening the fully human antibody response against antigens.

In addition, another aspect of the invention relates to the design ofthe desired non-endogenous V region (e.g., human). In particular, anentire V domain repertoire, or a portion thereof, may be incorporatedinto the genome of the cell, or a tailored V domain repertoire may beincorporated. For example, in certain embodiments it is preferred toomit V domain gene segments that are missing from some human haplotypesand instead tailoring the V domain repertoire to be composed of only thefunctional V gene segments common across all known human haplotypes.Doing so provides antibody drug candidates with V domains that arebetter immune tolerized across all potential patients, therebypreventing the induction of a dangerous immune response uponadministration of the encoded antibody to a subject. One or more Vdomain gene segments may be incorporated into the genome of a cell.

In certain embodiments of the invention, constructs containing thedesired Ig loci gene segments are used to incorporate the geneticinformation into the target cell genome via homologous recombination. Inparticular, the nature of BAC engineering in E. coli provides additionalopportunities to finely tailor the immunoglobulin loci prior tointroduction into competent cells. BAC libraries and the completesequence of the Ig loci are available for many species. Syntheticconstructs can also be finely tailored as described herein.

The ability to finely tailor the constructs described herein providesthe ability to introduce specific non-endogenous and syngeneiccomponents. For example, the non-endogenous DH cluster can be replacedor supplemented with D genes from other species, such as from non-humanprimate, rabbit, rat, camelid, hamster etc. D gene segments within theIgH loci can be defined from publicly available sequence or geneticstructure information, or by testing using appropriate D specific probesor primers. The orthologous D gene clusters or portions thereof can behomologously recombined into the constructs or assembled in silico andthen synthesized, therein replacing or adding to the cluster ofnon-endogenous D gene segments.

Because of the significant diversification that occurs in making thecomplementarity determining region-3 (CDR3) and because the structure ofthe V region is such that the CDR3 is relatively solvent inaccessible,immunogenicity to the CDR3 sequence is of less concern. Therefore, aminoacids encoded by non-human D genes incorporated into the CDR3 are lesslikely to be immunogenic upon administration to a human. D genes derivedfrom another species could confer an advantage by producing novel CDR3structures that would expand the range of epitope specificities andaffinities in a panel of antigen-specific antibodies, therein broadeningthe quality of activities mediated by a panel of mAbs.

Similarly, the JH gene cluster, i.e., one or more JH gene segments, canbe from a different non-endogenous species due to the relative sequenceconservation across mammals. The JH gene segment may be derived from anyanimal, e.g., human, non-human primate, rabbit, sheep, rat, hamster,camelid and mouse. In particular embodiments, the JH gene segment ishuman.

In further aspects, after engineering the Ig loci constructs, they areintroduced into non-human mammalian cells and are randomly integratedinto the genome. Methods for introducing one or more constructscomprising the altered Ig locus, or portion thereof, include, forexample, electroporation, lipofection, calcium phosphate precipitation,E. coli spheroplast fusion, yeast spheroplast fusion and microinjection,either into the pronucleus of a fertilized egg to make transgenicanimals directly or into cells cultured in vitro. In certainembodiments, the construct is engineered to carry a selectable markergene, e.g., G418^(R), hygromycin^(R), puromycin^(R), 5′ of the most 5′ Vgene. A selectable marker gene may also be 3′ of the most 5′ V gene. Theselectable marker gene may be flanked by site-specific recombinaserecognition sequences, which if brought into the presence ofrecombinase, will recombine and delete the intervening selectablemarker. This is particularly important if a selectable marker cassetteis located 3′ of the most 5′ V gene and near an enhancer sequence so asto not attenuate the function of the enhancer.

In certain embodiments, two or more constructs, such as BACs, areintroduced into the cell in a single step. If two constructs areintroduced into the cell simultaneously, they will typicallyco-integrate. Some of the co-integrated constructs will integrate in afunctional head-to-tail fashion with, for example, V, (D) and J segmentsoperably oriented 5′ of C region gene segments. Co-introduced constructscan be any combination of BACs, YACs, plasmids, bacteriophage, P1s etc.In some instance, there will be a single-copy integration of the twoconstructs, creating a single-copy transgene. In other instances, therewill be a multi-copy integration of the two constructs. Multi-copyintegration is not necessarily undesirable as it can yield beneficialconsequences, such as increased expression of the transgene, resultingin more of the desired gene product. However, if a single copy of thetransgene is desired, there are in vitro and in vivo processes for doingso.

For instance, if a site-specific recombinase sequence is positioned atthe 3′ end of the 5′ construct and if a site-specific recombinasesequence is positioned at the 5′ end of the 3′ construct, resultingco-integrants of the 5′ construct and the 3′ construct both oriented inthe 5′ to 3′ manner will have site-specific recombinase sequencesoriented so that any intervening sequence between the terminal 5′construct and the terminal 3′ construct would be deleted upon exposureto the site-specific recombinase, and thus the terminal 5′ constructbecomes operably linked to the terminal 3′ construct, resulting in asingle copy transgene. This process may be conducted either in vitro inculture mammalian cells or in vivo in transgenic animals expressing therecombinase (for example of resolving a multi-copy single constructtransgene into a single-copy transgene in vivo, see Janssens et al.Proc. Natl. Acad. Sci. (2006) 103: 15130-15135.) Functional transgenescan also be made by pronuclear co-microinjection of 3 or more constructs(see US Patent Application Publication No. 2010/0077497.)

After introducing the Ig locus or loci described herein into the genomeof a cell to replace (e.g., functionally replace) an endogenous Iglocus, or portion thereof, a non-human animal can be produced. If thenon-human mammalian cells are embryonic stem cells, geneticallyengineered non-human mammals, such as mice and rats, can be producedfrom the cells by methods such as blastocyst microinjection followed bybreeding of chimeric animals, morula aggregation. If the cells aresomatic cells, cloning methodologies, such as somatic cell nucleartransfer, can be used to produce a transgenic animal. Multi-stagebreeding is used to produce animals heterozygous or hemizygous formodified IgH and IgL loci (either Igκ or Igλ, or both Igκ and Igλ). Micewith modified IgH and IgL loci can be further bred to produce micehomozygous for IgH and IgL (either Igκ or Igλ, or both Igκ and Igλ).

The engineered Ig loci described herein will function in the non-humananimals. By using appropriate detection reagents, e.g., anti-human CH1domain antibodies or anti-human CL antibodies, it is possible to detectthe antibodies produced by the engineered locus even in the presence ofantibodies expressed from an active endogenous locus. Furthermore, in amouse, for example, it is possible to use allotypic sequences in themouse portion of the constant region of the transgene that are differentfrom the allotypic sequence of the constant region of the recipientmouse strain, e.g., mouse IgH a allotypes (Balb/c) versus IgH ballotypes (C57BL/6).

Inactivation of Endogenous Ig Loci

In certain embodiments, it may be desirable to functionally inactivateone or more of the endogenous Ig loci in the recipient non-human mammal.Various methods known in the art can be used to inactivate theendogenous Ig loci. An animal comprising an engineered Ig transgene isbred with an animal comprising one or more inactivated endogenous locito derive an animal capable of expressing antibodies from the Igtransgene and without production of the complete native immunoglobulinfrom the inactivated endogenous loci with the Ig transgene thereinfunctionally replacing the inactivated endogenous locus.

The very fine tailoring of DNA sequences by combining in silicorecombination with in vitro DNA synthesis and assembly technologiesallows for the precise deletion and/or modification of the homologoustarget sequences. For instance, recombination signal sequences or splicedonor sequences for specific gene segments, e.g., J gene, may be alteredor deleted.

Components of an IgH locus that may be altered to down modulate and/orabrogate locus function include the JH cluster (complete deletion,removal of recombination signal sequences (RSS), splice donor sequencesor all or some of the above) (see, for example, U.S. Pat. No.5,939,598), Eμ, Cμ and Cδ, the D gene cluster (complete deletion,removal of recombination signal sequences (RSS), splice donor sequencesor all or some of the above), the VH genes (complete deletion, removalof recombination signal sequences (RSS), splice donor sequences or allor some of the above), and all of the constant region genes. Placing astrong constitutive promoter such as PGK in the position of criticalenhancer elements such as E.p can have severe deleterious consequenceson locus function, effectively bringing about inactivation.

Components of an Igκ locus that may be altered to down modulate and/orabrogate locus function include the Jκ cluster (complete deletion,removal of recombination signal sequences (RSS), splice donor sequencesor all or some of the above), Eκ, Cκ, and the Vκ genes (completedeletion, removal of recombination signal sequences (RSS), splice donorsequences or all or some of the above).

Components of the Igλ locus that may be altered to down modulate and/orabrogate locus function include the Jλ cluster (complete deletion,removal of recombination signal sequences (RSS), splice donor sequencesor all or some of the above), Eλ, Cλ, and the Vλ genes (completedeletion, removal of recombination signal sequences (RSS), splice donorsequences or all or some of the above). Deletion of larger sequenceunits such as the entire Igλ locus, the entire VH gene repertoire of theIgH locus etc. may be effected by serial insertion of site-specificrecombination sequences (lox P or frt) adjacent to the 5′ and 3′ ends ofthe sequence to be deleted followed by transient expression of therelevant recombinase, e.g., CRE or FLP. Various methods known in the artcan be used to inactivate the endogenous Ig loci. See for example: ChenJ., et al. Int Immunol. 1993 Jun.; 5(6):647-56; Jakobovits et al., Proc.Natl. Acad. Sci. (1993) 90: 2551-2555; Nitschke et al. Proc. Natl. Acad.Sci. (1993) 90: 1887-1891; U.S. Pat. No. 5,591,669; Afshar et al., J.Immunol. (2006) 176: 2439-2447; Perlot et al. Proc. Natl. Acad. Sci.(2005) 97: 14362-14367; Roes and Rajewsky J. Exp Med. (1993) 177: 45-55;Lutz et al. Nature (1998) 393: 797-801; Ren et al. Genomics (2004) 84:686-695; Zou et al. EMBO J. (1993) 12: 811-820; Takeda et al. EMBO J.(1993) 12: 2329-2336; Chen et al. EMBO J. (1993) 12: 821-830; Zou etal., J. Immunol. (2003) 170: 1354-1361; Zheng et al. Molec. Cell. Biol.(2000) 20: 648-655; Zhu et al. Proc. Natl. Acad. Sci. (2000) 97:1137-1142; Puech et al. Proc. Natl. Acad. Sci. (2000) 97: 10090-10095;LePage et al. Proc. Natl. Acad. Sci. (2000) 97: 10471-10476; Li et al.Proc. Natl. Acad. Sci. (1996) 93: 6158-6162.

In some embodiments, multiple deletions or multiple mutations areintroduced into an endogenous Ig locus to inactivate the endogenousimmunoglobulin locus, thereby solving the problem of partiallyinactivating an endogenous Ig locus. In particular, the two or moremutations independently impair both the formation of a functionalvariable domain and the formation of a constant region capable ofdriving primary B cell development. The mutations impair primary B celldevelopment because the resulting Ig sequence prevent formation of anIgH capable of mediating signal transduction (by itself or inassociation with Igα and/or Igβ), e.g., gene rearrangement is blocked,transcription or translation of a complete product fails, or the productcannot signal.)

In one instance, the endogenous J genes, Eμ, Cμ and Cδ are deleted. Inanother instance, the endogenous J genes, Eμ, Cμ and Cδ are all replacedwith a single drug-resistance cassette that is transcriptionally activein ES cells and B cells. An example of a drug-resistance cassette foruse in mice is the PGK-G418 neomycin resistance cassette comprising themouse pgk-1 promoter. Taken together, this deletion blocks V-D-Jrecombination (J deletion and Eμ replacement by an active expressioncassette) and primary B cell receptor signaling (deletion of CG and Cδ)and anchoring in the B cell membrane. The inactivation of multiplecomponents produces multiple layers of redundancy for inactivating theIgH locus at different developmental stages, creating a failsafe againstany one residual activity rescuing B cell development.

Other combinations of deletions or mutations can also be performed. Forexample, the entire C gene cluster may be deleted in combination with aJH deletion. Deletion of the entire D gene cluster in combination withCμ and Cδ would also be effective. Any combination of one or moremutations are contemplated herein as long as the resulting mutationsimpair formation of a functional variable region and formation of amembrane-anchored heavy chain constant region capable of signaltransduction, either directly or in combination with the accessorysignal transducing proteins Igα and/or Igβ.

Not all of each module needs to be deleted. For instance, a portion ofJ, Cμ or Cδ genes may be left in the immunoglobulin locus so long as oneor more cis regulatory elements such as recombination signal sequences(RSS), splice donor, and splice acceptor sequences are deleted ormutated or the formation of a functional open reading frame is obviated.Current methodologies for mutating or synthesizing precise DNA sequencesenable the creation of very specific, even single nucleotide, mutationsto be introduced. This provides the benefit of allowing for optimalpositioning of the DNA arms driving homologous recombination in ES cellswhile still inactivating the locus.

Deletion of portions of the IgH locus can be made in cells usinghomologous recombination techniques that are now standard for geneticengineering. Deletions may be made in one step or in multiple steps, andthey may be generated using one or more constructs. The deletions couldalso be made using site-specific recombinase systems such as Cre-lox orFlp-Frt. A combination of homologous recombination and site-specificrecombinase systems may be used. Other systems such as engineeredzinc-finger nucleases injected into fertilized eggs may be used toengineer deletions into the genes, in one or more steps to build up thenumber of deleted or mutated modules of the IgH locus.

A similar strategy may be employed for inactivating the endogenousimmunoglobulin kappa light chain and/or lambda light chain. Theimportant modules for inactivation are conserved, particularly the J, E(intronic enhancer) and C regions, between all of the Ig loci. Inembodiments regarding the inactivation of an Ig light chain locus,inactivation of the constant region will prevent the formation of acomplete antibody molecule or a Fab domain in that the light chainconstant region is unable to form a disulfide bond with the heavy chain.

For example, an endogenous Igκ locus can be inactivated by replacing theJ genes, Eκ, and Cκ with a single drug-resistance cassette that istranscriptionally active in ES cells and B cells, such as the PGK-G418neomycin resistance cassette. Taken together, this deletion blocks V-Jrecombination (J deletion and Eκ replacement by an active expressioncassette) and pairing of an Igκ light chain with a heavy chain in anantibody (deletion of Cκ). Any combination of one or more mutations arecontemplated herein as long as the resulting mutations impair formationof a functional variable region and formation of a light chain constantregion capable of forming a disulfide bond with a heavy chain.

In another embodiment, all of the J-C pairs of the Ig, locus 3′ of theVλ gene segment can be deleted using a site-specific recombinase system,such as Cre/loxP. The deletion of all of the J segment genes preventsV-J rearrangement, and therefore impairs the formation of a functionalvariable region. The deletion of the Cλ gene segments prevents theformation of a functional constant region, thereby preventing theformation of a constant region capable of forming a disulfide bond withany IgH chain. In yet another embodiment, inactivation of the mouse Ig,is achieved through two separate inactivations. The first isinactivation is of Vλ1 and the second is inactivation is of both Vλ2 andVλx. The second inactivation may be done before the first inactivation.Inactivation may be achieved through inactivation of RSS 5′ or 3′ ofeach of the Vλ genes, inactivation of the promoters 5′ of each gene, orinactivation of the coding sequences. The inactivation may be throughmutation, either point, insertion or deletion, to render non-functional,or complete deletion.

The endogenous Ig locus of a non-human cell may be inactivated byhomologous recombination with one or more constructs designed tointroduce the deletions or mutations capable of impairing both theformation of a functional variable domain and the formation of aconstant region capable of driving primary B cell development. Methodsfor effecting homologous recombination in mouse and rat ES cells areknown in the art. Upon homologous recombination between the flankingregions located on the construct and the corresponding homologousendogenous DNA sequences in the cell, the desired deletions or mutationsare incorporated into the endogenous Ig locus.

Cells that have undergone a correct recombination event can be screenedfor using positive and negative selection markers, such as drugresistance. To further confirm homologous recombination, genomic DNA isrecovered from isolated clones and restriction fragment lengthpolymorphism (RFLP) analysis performed by a technique such as Southernblotting with a DNA probe from the endogenous loci, said probe mappingoutside the replaced region. RFLP analysis shows allelic differencesbetween the two alleles, the endogenous DNA and incoming DNA, when thehomologous recombination occurs via introduction of a novel restrictionsite in the replacing DNA.

Various assays known in the art, including, but not limited to, ELISAand fluorescence microscopy, may be used to confirm that the mutationsintroduced into the endogenous Ig locus impair the expression of afunctional Ig heavy or light chain by the cell. An absence of theendogenous Ig heavy or light chain indicates that its expression isimpaired. Other well known assays, such as RT-PCR, can determine whetheror not the modified locus is able to be transcribed.

Cells having one or more inactivated Ig loci may be used to producetransgenic non-human animals, e.g., mice, that have one or moreinactivated Ig loci. After engineering the mutated Ig locus intonon-human cells to delete or replace portions of the endogenous Ig loci,genetically engineered non-human mammals, such as mice, can be producedby now-standard methods such as blastocyst microinjection followed bybreeding of chimeric animals, morula aggregation or cloningmethodologies, such as somatic cell nuclear transfer.

Breeding Strategies

Certain embodiments provide a method of producing a non-human mammalhaving a genome encoding non-endogenous VH and CH1 gene segments and anon-endogenous Ig light chain locus comprising the steps of breeding anon-human mammal comprising a chimeric Ig heavy chain locus, wherein theIg heavy chain locus comprises the non-endogenous VH and CH1 genesegments, with a non-human mammal comprising a non-endogenous Ig lightchain locus; selecting offspring having a genome comprising the chimericIg heavy chain locus and the non-endogenous Ig light chain locus;further breeding the offspring; and producing offspring having a genomehomozygous for the chimeric heavy and non-endogenous light chain loci.In related embodiments, the genome of the mammal also encodes anon-endogenous JH gene segment.

Further embodiments comprise selecting offspring having a genomecomprising the chimeric Ig heavy chain locus and the non-endogenous Iglight chain locus; further breeding the offspring with non-human mammalshaving functionally inactivated endogenous Ig loci; and producingoffspring and further breeding to produce offspring having a genomehomozygous for functionally inactivated endogenous Ig loci, the chimericheavy and the non-endogenous light chain loci. In related embodiments,the genome of the mammal also encodes a non-endogenous JH gene segment.

The genetic engineering strategies described herein can be applied toengineering of mice and other animals so as to express non-endogenoussequence V regions coupled with xenogeneic C regions, or completelynon-endogenous antibodies, or some intermediate thereof. For animals forwhich there is a current lack of ES cell technology for geneticengineering through blastocyst microinjection or morula aggregation, theendogenous loci can be modified in cells amenable to various cloningtechnologies or developmental reprogramming (e.g., induced pluripotentstem cells, IPS). The increased frequency of homologous recombinationprovided by the BAC technology provides the ability to find doublyreplaced loci in the cells, and cloned animals derived therefrom wouldbe homozygous for the mutation, therein saving time and costs especiallywhen breeding large animals with long generation times. Iterativereplacements in the cultured cells could provide all the requisiteengineering at multiple loci and then direct production of animals usingcloning or IPS technology, without cross-breeding, to produce theappropriate genotype. The ability to finely tailor the introduced Iggenes and also finely specify the sites into which they are introducedprovides the ability to engineer enhancements that provide betterfunction. Engineered animals such as goats, bovines, ovines, equines,rabbits, llamas, dogs etc. are a source of fully human polyclonalantibodies.

Furthermore, if BACs are engineered in E. coli with DNA componentsrequired for chromosome function, e.g., telomeres and a centromere,preferably, but not required, of the recipient species for optimalfunction, e.g., mouse telomeres and a mouse centromere, they can beintroduced into the recipient cell by electroporation, microinjectionetc. and function as artificial chromosomes. These BAC-based artificialchromosomes also can be used as a foundation for subsequent rounds ofhomologous recombination for building up larger artificial chromosomes.

The engineered Ig locus or loci described herein provided on vectorssuch as plasmids, BACs or YACs can also be used as standard transgenesintroduced via microinjection into the pronucleus of an embryo such asmouse, rabbit, rat, or hamster. Several BACs, YACs, plasmids or anycombination thereof can be co-microinjected and will co-integrate tomake a functional locus. Various methods known in the art and describedherein can be used to inactivate the endogenous Ig loci and the animalswith an engineered Ig transgene bred with those with one or moreinactivated endogenous loci to derive genotypes expressing antibodiesfrom the transgene and without production of the complete nativeimmunoglobulin from the inactivated endogenous loci.

Antibodies

A chimeric antibody, or antigen-binding fragment thereof, as disclosedherein comprises a non-endogenous variable domain and a chimeric heavychain constant region. In particular, the chimeric heavy chain constantregion comprises a non-endogenous CH1 domain. In certain embodiments,the chimeric antibody comprises a chimeric heavy chain and anon-endogenous light chain. In other embodiments, the chimeric antibodycomprises a chimeric heavy chain and an endogenous light chain. In oneembodiment, the chimeric heavy chain variable region is encoded bypolynucleotide sequences derived from two or more non-endogenousspecies.

In certain embodiments, the chimeric heavy chain comprises anon-endogenous upper hinge region. In a related embodiment, the chimericheavy chain comprises non-endogenous upper and middle hinge regions.

Eukaryotic Transgenes Comprising Sequences Designed in Silico and MadeSynthetically

The ability to obtain sequences, for genes, loci and full genomes, andtranscriptome sequences, either from public databases with annotations,or derived using commercially available sequencing technology, orderived through commercial operation performing sequencing on acontractual basis, means that DNA sequences can be readily manipulatedin silico, e.g., taken apart and reassembled, either within genes orloci, or between genes or loci, across the same species, differentstrains of a species, or across two or more different species.Heretofore eukaryotic transgenes, particularly metazoans, have beenconstructed from DNAs derived from a natural source. These naturalsource DNAs include genomic DNA libraries cloned into various vectorssuch plasmid, bacteriophage, P1s, cosmids, BACs, YACs and MACs and cDNAlibraries cloned into vectors, generally plasmids or bacteriophage.Methodologies for recombining DNAs carried on these vectors and forintroducing small alterations such as site-directed mutations are wellknown in the art and have been deployed to make transgenes composed ofsequence that overall conforms to the sequence of the parental DNA inthe library from which they were isolated. In some instances, portionsof DNA are missing from the library, or a library from the desiredanimal, strain or haplotype thereof may be unavailable and not able tobe constructed using ordinary skill in the art.

For example, there may be preferred allelic variants to be included in atransgene and said allelic variant is not available in any library and,furthermore, source nucleic acid such as RNA or DNA may not beavailable. In complex loci with many genes or exons and cis regulatoryelements, it can be technically infeasible to procure and recombine intoone transgenes the DNAs encoding such if they are from different speciesand strains or haplotypes. Thus, the means by which to create DNA ofcomplexly engineered, particularly from a completely in silico design,is not possible using standard methodologies.

Synthetic means of creating DNA sequences have been described, arecommercially available and can be used to make DNA sequences based on acompletely in silico design. However, whether they can be introducedand, in particular, expressed in eukaryotes, particularly metazoans, hasheretofore been unknown.

Some of the Ig transgene constructs disclosed herein comprise suchcomplex sequence composition that they cannot be engineered to thedesired precision and accuracy by previously described means. Furthercontemplated transgene structures include a germline configured DNA inwhich are replaced only coding sequences, all or a part thereof, bynon-endogenous coding sequences so that all of the cis regulatorysequences are endogenous, retaining completely native gene regulationoptimal for, position-independent, copy-number dependent,tissue-specific, developmental-specific gene regulation, e.g, an IgHsequence which comprises completely mouse DNA except for sequencesencoding human VH, DH, JH, CH1 and upper hinge sequences replacing theirmouse orthologues; an FcγR sequence which comprises completely mouse DNAexcept for sequences encoding human FcγR replacing their mouseorthologues; an IgH sequence which comprises completely mouse DNA exceptfor sequences encoding camelid VH, DH, JH, sequences replacing theirmouse orthologues; an IgH sequence which comprises completely mouse DNAexcept for sequences encoding human VH, JH, CH1 and upper hingesequences replacing their mouse orthologues and non-human, non-mouse DHcoding sequences, e.g., rabbit, replacing their mouse orthologues; anIgH sequence which comprises completely mouse DNA except for sequencesencoding VH, DH, JH, from a species relevant to animal healthcare, e.g.,canine, feline, ovine, bovine, porcine, replacing their mouseorthologues; an IgL sequence which comprises completely mouse DNA exceptfor sequences encoding human VL, JL, and optionally CL, replacing theirmouse orthologues; an IgL sequence which comprises completely mouse DNAexcept for sequences encoding camelid VL, JL, and optionally CL,replacing their mouse orthologues; an IgL sequence which comprisescompletely mouse DNA except for sequences encoding VL, JL, andoptionally CL from a species relevant to animal healthcare, e.g.,canine, feline, ovine, bovine, porcine, replacing their mouseorthologues. Other examples include deleting unneeded or undesirable DNAsequences, e.g., V genes that are pseudogenes, V genes that produceproducts that can misfold, V genes that are absent from some humanhaplotypes, large tracts of non-regulatory DNA, CH genes nottherapeutically important. Other examples include altering DNA sequencesfor optimizing transgene function or producing a desired producttherefrom, e.g., using the most prevalent allele of a V gene, repairingthe mouse Igλ 3′ enhancer to restore NFκb binding. Transgenes maycomprise parts that are synthetic and parts that are from naturalsources. DNAs for inactivating genes may also comprise synthetic DNA,all or in part. Moreover, the method for transgene constructiondescribed herein is not limited to immunoglobulin loci. Any transgenecan be constructed by the steps of first using in silico methods torecombining and assemble the sequence from various DNA sequence andsecond of employing available synthetic DNA methods to create thephysical DNA.

Methods of Producing Antibodies

An animal carrying the modified locus or loci can be immunized with anantigen using various techniques available in the art. Antigens may beselected for the treatment or prevention of a particular disease ordisorder, such as various types of cancer, graft versus host disease,cardiovascular disease and associated disorders, neurological diseasesand disorders, autoimmune and inflammatory disorders, and pathogenicinfections. In other embodiments, target antigens may be selected todevelop an antibody that would be useful as a diagnostic agent for thedetection one of the above diseases or disorders.

Antigen-specific repertoires can be recovered from immunized animals byhybridoma technology, single-cell RT-PCR for selected B cells, byantibody display technologies, and other methods known in the art. Forexample, to recover human/mouse chimeric mAbs from mouse-derivedhybridomas, a human V-CH1-mouse hinge+CH2+CH3 antibody or a humanV-CH1-upper/middle hinge-mouse lower hinge+CH2+CH3 antibody (dependingupon the IgH locus engineering) is secreted into the culture supernatantand can be purified by means known in the art such as columnchromatography using protein A, protein G, etc. The purified antibodycan be used for further testing and characterization of the antibody todetermine potency in vitro and in vivo, affinity etc.

In addition, since they can be detected with an antibody specific forthe endogenous constant region used as a secondary agents, a human V-CH1(upper/middle hinge)-non-human CH2-CH3 mAb may be useful forimmunochemistry assays of human tissues to assess tissue distributionand expression of the target antigen. This feature of the chimericantibodies of the present invention allows for specificity confirmationof the chimeric mAb over fully human mAbs because of occasionalchallenges in using anti-human constant region secondary detectionagents against tissues that contain normal human Ig and from the bindingof human Fc regions to human FcR expressed on cells in some tissues.

The non-endogenous variable regions of the mAbs can be recovered andsequenced by standard methods. Either before or after identifying leadcandidate mAbs, the genes, either genomic DNA or cDNAs, for thenon-endogenous VH and VL domains can be recovered by various molecularbiology methods, such as RT-PCR, and then appended to DNA encoding theremaining portion of the non-endogenous constant region, therebyproducing a fully non-endogenous mAb. For example, a fully human mAb maybe generated. The DNAs encoding the now fully non-endogenous VH-CH andnon-endogenous VL-CL would be cloned into suitable expression vectorsknown in the art or that can be custom-built and transfected intomammalian cells, yeast cells such as Pichia, other fungi etc. to secreteantibody into the culture supernatant. Other methods of production suchas ascites using hybridoma cells in mice, transgenic animals thatsecrete the antibody into milk or eggs, and transgenic plants that makeantibody in the fruit, roots or leaves can also be used for expression.The fully non-endogenous recombinant antibody can be purified by variousmethods such as column chromatography using protein A, protein G etc.

A purified antibody can be lyophilized for storage or formulated intovarious solutions known in the art for solubility and stability andconsistent with safe administration into animals, including humans.Purified recombinant antibody can be used for further characterizationusing in vitro assays for efficacy, affinity, specificity, etc., animalmodels for efficacy, toxicology and pharmacokinetics etc. Further,purified antibody can be administered to humans and non-human animalsfor clinical purposes such as therapies and diagnostics for disease.

Various fragments of the non-endogenous V-CH1-(upper/middlehinge)-endogenous CH2-CH3 mAbs can be isolated by methods includingenzymatic cleavage, recombinant technologies, etc. for various purposesincluding reagents, diagnostics and therapeutics. The cDNA for therepertoire of non-endogenous variable domains+CH1 or just thenon-endogenous variable domains can be isolated from the engineerednon-human mammals described above, specifically from RNA from secondarylymphoid organs such as spleen and lymph nodes, and the VH and VL cDNAsimplemented into various antibody display systems such as phage,ribosome, E. coli, yeast, mammalian etc. The transgenic mammals may beimmunologically naïve or optimally may be immunized against an antigenof choice. By using appropriate PCR primers, such as 5′ in the leaderregion or framework 1 of the variable domain and 3′ in the human CH1 ofCγ genes, the somatically matured V regions can be recovered in order todisplay solely the affinity-matured repertoire. The displayed antibodiescan be selected against the target antigen to efficiently recoverhigh-affinity antigen-specific Fv or Fabs, and are void of theendogenous CH2-CH3 domains that would be present if mAbs were recovereddirectly from the mammals. Moreover, it is not necessary that theanimals carrying the IgH and IgL transgene be functionally inactivatedfor the endogenous Ig loci. Animals heterozygous for IgH and IgL loci,or animals carrying the IgH and IgL transgenes and heterozygous forinactivated endogenous IgH and IgL loci, which produce the chimericantibodies described herein as well as both fully-endogenous antibodies(e.g., mouse antibodies) and mixed endogenous and non-endogenousantibodies (e.g., human-mouse antibodies), can also be used to generateantigen-specific non-endogenous V-endogenous C mAbs (e.g., human V-mouseC mAbs). Animals carrying just one Ig transgene, e.g., IgH, could beused as a source of non-endogenous (e.g., human) VH domains (VH-CH1) andother animals carrying just one different Ig transgene, e.g., IgL,either Igκ or Igλ, could be used as a source of non-endogenous (e.g.,human) VL domains (VL-CL) and then the VH-CH1 and VL-CL sequencescombined into an antibody display library to display fully humanantibodies. In such animals, both, one or none of the endogenous Ig locimay be activated. The animals may be immunized so as to enable recoveryof affinity-mature VH and VL. For example, an antibody display libraryfrom two separate mice—one with human VH-CH1-mouse CH2-CH3 and the otherwith human Vκ-Cκ—could be used to recover fully human antibodies usingwell-established techniques in molecular biology.

Methods of Use

Purified antibodies of the present invention may be administered to asubject for the treatment or prevention of a particular disease ordisorder, such as various types of cancer, graft versus host disease,cardiovascular disease and associated disorders, neurological diseasesand disorders, autoimmune and inflammatory disorders, allergies, andpathogenic infections. In preferred embodiments, the subject is human.

Antibody compositions are administered to subjects at concentrationsfrom about 0.1 to 100 mg/ml, preferably from about 1 to 10 mg/ml. Anantibody composition may be administered topically, intranasally, or viainjection, e.g., intravenous, intraperitoneal, intramuscular,intraocular, or subcutaneous. A preferred mode of administration isinjection. The administration may occur in a single injection or aninfusion over time, i.e., about 10 minutes to 24 hours, preferably 30minutes to about 6 hours. An effective dosage may be administered onetime or by a series of injections. Repeat dosages may be administeredtwice a day, once a day, once a week, bi-weekly, tri-weekly, once amonth, or once every three months, depending on the pharmacokinetics,pharmacodynamics and clinical indications. Therapy may be continued forextended periods of time, even in the absence of any symptoms.

A purified antibody composition may comprise polyclonal or monoclonalantibodies. An antibody composition may contain antibodies of multipleisotypes or antibodies of a single isotype. An antibody composition maycontain unmodified chimeric antibodies, or the antibodies may have beenmodified in some way, e.g., chemically or enzymatically. An antibodycomposition may contain unmodified human antibodies, or the humanantibodies may have been modified in some way, e.g., chemically orenzymatically. Thus an antibody composition may contain intact Igmolecules or fragments thereof, i.e., Fab, F(ab′)2, or Fc domains.

Administration of an antibody composition against an infectious agent,alone or in combination with another therapeutic agent, results in theelimination of the infectious agent from the subject. The administrationof an antibody composition reduces the number of infectious organismspresent in the subject 10 to 100 fold and preferably 1,000 fold, andmore than 1,000 fold.

Similarly, administration of an antibody composition against cancercells, alone or in combination with another chemotherapeutic agent,results in the elimination of cancer cells from the subject. Theadministration of an antibody composition reduces the number of cancercells present in the subject 10 to 100 fold and preferably 1,000 fold,and more than 1,000 fold.

In certain aspects of the invention, an antibody may also be utilized tobind and neutralize antigenic molecules, either soluble or cell surfacebound. Such neutralization may enhance clearance of the antigenicmolecule from circulation. Target antigenic molecules for neutralizationinclude, but are not limited to, toxins, endocrine molecules, cytokines,chemokines, complement proteins, bacteria, viruses, fungi, andparasites. Such an antibody may be administered alone or in combinationwith other therapeutic agents including other antibodies, otherbiological drugs, or chemical agents.

It is also contemplated that an antibody of the present invention may beused to enhance or inhibit cell surface receptor signaling. An antibodyspecific for a cell surface receptor may be utilized as a therapeuticagent or a research tool. Examples of cell surface receptors include,but are not limited to, immune cell receptors, adenosine receptors,adrenergic receptors, angiotensin receptors, dopamine and serotoninreceptors, chemokine receptors, cytokine receptors, histamine receptors,etc. Such an antibody may be administered alone or in combination withother therapeutic agents including other antibodies, other biologicaldrugs, or chemical agents.

It is also contemplated that an antibody of the present invention may befurther modified to enhance therapeutic potential. Modifications mayinclude direct- and/or indirect-conjugation to chemicals such aschemotherapeutic agents, radioisotopes, siRNAs, double-stranded RNAs,etc. Other modifications may include Fc regions engineered for eitherincreased or decreased antibody-dependent cellular cytotoxicity, eitherincreased or decreased complement-dependent cytotoxicity, or increasedor decreased circulating half-life.

In other embodiments, an antibody may be used as a diagnostic agent forthe detection one of the above diseases or disorders. A chimericantibody may be detected using a secondary detection agent thatrecognizes a portion of the antibody, such as an Fc or Fab domain. Inthe case of the constant region, the portion recognized may be a CH1,CH2, or a CH3 domain. The Cκ and Cλ domain may also be recognized fordetection. Immunohistochemical assays, such as evaluating tissuedistribution of the target antigen, may take advantage of the chimericnature of an antibody of the present invention. For example, whenevaluating a human tissue sample, the secondary detection agent reagentrecognizes the non-human portion of the Ig molecule, thereby reducingbackground or non-specific binding to human Ig molecules that may bepresent in the tissue sample.

Pharmaceutical Compositions and Kits

The present invention further relates to pharmaceutical compositions andmethods of use. The pharmaceutical compositions of the present inventioninclude an antibody, or an antigen-binding fragment thereof, in apharmaceutically acceptable carrier. Pharmaceutical compositions may beadministered in vivo for the treatment or prevention of a disease ordisorder. Furthermore, pharmaceutical compositions comprising anantibody, or an antigen-binding fragment thereof, of the presentinvention may include one or more agents for use in combination, or maybe administered in conjunction with one or more agents.

The present invention also provides kits relating to any of theantibodies, or antigen-binding fragments thereof, and/or methodsdescribed herein. Kits of the present invention may be used fordiagnostic or treatment methods. A kit of the present invention mayfurther provide instructions for use of a composition or antibody andpackaging.

A kit of the present invention may include devices, reagents, containersor other components. Furthermore, a kit of the present invention mayalso require the use of an apparatus, instrument or device, including acomputer.

EXAMPLES

The following examples are provided as further illustrations and notlimitations of the present invention. The teachings of all references,patents and published applications cited throughout this application, aswell as the Figures are hereby incorporated by reference.

Example 1 Construction of BAC C5P12

A BAC vector is based on the F-factor found in E. coli. The F-factor andthe BAC vector derived from it are maintained as low copy plasmids,generally found as one or two copies per cell depending upon its lifecycle. Both F-factor and BAC vector show the fi+ phenotype, whichexcludes an additional copy of the plasmid in the cell. By thismechanism, when E. coli already carries and maintains one BAC, and thenan additional BAC is introduced into the E. coli, the cell maintainsonly one BAC, either the BAC previously existing in the cell or theexternal BAC newly introduced. This feature is extremely useful forselectively isolating BACs homologously recombined as described below.

The homologous recombination in E. coli requires the functional RecAgene product. In this example, the RecA gene had a temperature-sensitivemutation (recA^(ts)) so that the RecA protein was only functional whenthe incubation temperature was below 37° C. When the incubationtemperature was above 37° C., the Rec A protein was non-functional orhad greatly reduced recombination activity. This temperature sensitiverecombination allowed manipulation of RecA function in E. coli so as toactivate conditional homologous recombination only when it was desired.It is also possible to obtain, select or engineer cold-sensitivemutations of Rec A protein such that the protein is only functionalabove a certain temperature, e.g., 37° C. In that condition, the E. coliwould be grown at a lower temperature, albeit with a slower generationtime, and recombination would be triggered by incubating at above 37° C.for a short period of time to allow only a short interval ofrecombination.

Homologous recombination in E. coli was carried out by providingoverlapping DNA substrates that are found in two circular BACs. BAC P12(California Institute of Technology BAC library) was 182 kb in totalsize of which 172 kb was an insert of human genomic DNA comprising humanVκ genes (from IGKV1-5 to IGKV1-12, FIG. 1). BAC P12 was carried bypBeloBAC2 vector that had a zeocin resistant gene Zeo^(R). BAC C5(California Institute of Technology BAC library) carried a kanamycinresistance transposon cassette (kan^(R)) for selection in E. coli, KAN-2(Epicentre Biotechnologies). BAC C5 was 225 kb in total size of which218 kb was an insert of human genomic DNA comprising human Vκ genes(from IGKV4-1 to IGK1-6), the Jκ cluster, Cκ and 3′ regulatory elements.BACs C5 and P12 carried a 70 kb of homology in the insert DNA. BAC C5was carried in E. coli recAts.

Purified BAC P12 DNA was electroporated into E. coli recA^(ts) carryingBAC C5. The cells were incubated at 30° C., the permissive temperaturefor recA^(ts) activity, for 30 minutes. E. coli carrying homologousrecombinants of the two BACs (kan^(R)zeo^(R)) were selected by platingon plates of selective low salt LB medium (Invitrogen) containing zeocin(50 ug/ml) and kanamycin (25 ug/ml) and incubated at 40° C., anon-permissive temperature for the recA^(ts) activity. Homologousrecombinants in the 70 kb homology shared between C5 and P12 produced asingle BAC of 407 kb in total size, of which 320 kb represents therecombined inserts of C5 and P12 (FIG. 1). As expected, the homologousrecombination event created a duplication of the 70 kb overlap, one copyof which was situated between the repeated copies of the pBeloBAC vectorsequences and the other copy now joining the two fragments of human DNAfrom the Igκ locus into one contiguous segment (FIG. 1). E. colicolonies that grew on the double-selection plates exhibitedkan^(R)zeo^(R), were picked and BAC DNA isolated by miniprep. BAC DNAwas digested with NotI and run on pulse-field gels. Clones exhibited theexpected pattern of bands (FIG. 2, left BAC map and left gel photo).

The 70 kb repeat between the two copies of the pBeloBAC vector wasexcised by using CRE-recombinase acting on the two loxP sites that existon pBeloBAC (FIG. 2). Purified BAC (C5+P12) DNA was treated with CRErecombinase (New England Biolabs) in vitro according to themanufacturer's recommended conditions. The treated DNA was introducedinto a RecA deficient (recA⁻) strain of E. coli via electroporation andthe resulting bacteria plated on chloramphenicol (Cm) containing platesand incubated at 37° C. All of the pBeloBAC vectors carry Cm^(R) gene.The resolved BAC had lost the duplication of the 70 kb overlap and thesequence for pBeloBAC vector 2 (FIG. 2, right hand BAC map). Thecorrectly resolved BAC lost both markers of Zeo^(R) and Km^(R).

E. coli colonies that grew on plates exhibited Cm^(R), were picked andBAC DNA was isolated by miniprep. BAC DNA was digested with NotI and runon pulse-field gels. Clones exhibited the expected pattern of bands(FIG. 2, right BAC map and right gel photo). The resolved BAC (C5P12)was 327 kb in total size of which 320 kb is human genomic DNA from, in5′ to 3′ order, Vκ1-12 through the 3′ cis regulatory regions, including8 functional Vκ genes, the entire Jκ cluster and Cκ. To make the BAC(see, C5P12C20 in EXAMPLE 2), Tpn-Zeo was inserted at 15 kb from thejunction of the vector (FIG. 3). Tpn-Zeo was constructed by insertingZeo^(R) gene into Transposon Construction Vector (EpicentreBiotechnologies), pMOD-3<R6Kγori/MCS> plasmid.

Example 2 Construction of a 489 Kb BAC Comprising the Majority of theHuman Igk Locus

Homologous recombination in E. coli was carried out by providingoverlapping DNA substrates that are found in two circular BACs. BAC C20(California Institute of Technology BAC library) was 218 kb in totalsize of which 206 kb was an insert of human genomic DNA comprising humanVκ genes. BAC C20 carried the KAN-2 kanamycin resistance transposoncassette (kan^(R)) for selection in E. coli. BAC C5P12 made in Example 1carried a zeocin resistance transposon cassette (zeo^(R)) for selectionin E. coli. C20 and C5P12 carried a 44 kb of native homology in theinsert DNA. BAC C5P12 was carried in E. coli recA^(ts).

Purified BAC C20 DNA was electroporated into E. coli recA^(ts) carryingBAC C5P12. The cells were incubated at 30° C., the permissivetemperature for recA^(ts) activity, for 30 minutes. The fi+ phenotypeconferred by the pBeloBAC vector prohibited the maintenance of more thanone BAC in the cell, resulting in a population of E. coli carrying onlyC20 (kan^(R)zeo^(S)), only C5P12 (kan^(S)zeo^(R)), recombinants betweenthe two BACs (kan^(R)zeo^(R)) or no BAC (kan^(S)zeo^(S)). E. colicarrying homologous recombinants of the two BACs were selected byplating on plates of selective low salt LB medium (Invitrogen)containing zeocin (50 ug/ml) and kanamycin (25 ug/ml) and incubated at40° C., a non-permissive temperature for the recA^(ts) activity.

Homologous recombinants in the 44 kb homology shared between C20 andC5P12 produced a single BAC of 545 kb in total size (“C”), of which 482kb represents the recombined inserts of C20 and C5P12 (see FIG. 4). Asexpected, the homologous recombination event created a duplication ofthe 44 kb overlap, one copy of which was situated between the repeatedcopies of the pBeloBAC vector sequences and the other copy now joiningthe two fragments of human DNA from the Igκ locus into one contiguoussegment (FIG. 4).

E. coli colonies that grew on the double-selection plates exhibitedkan^(R)zeo^(R), were picked and BAC DNA isolated by miniprep. BAC DNAwas digested with NotI and run on pulse-field gels. Clones exhibited theexpected pattern of bands (FIG. 5, left BAC map and left gel photo).

The 44 kb repeat between the two copies of the pBeloBAC vector wasexcised by using CRE-recombinase acting on the two loxP sites that existon pBeloBAC (FIG. 5). Purified BAC C DNA was treated with CRErecombinase (New England Biolabs) in vitro according to themanufacturer's recommended conditions. The treated DNA was introducedinto a RecA deficient (recA⁻) strain of E. coli via electroporation andthe resulting bacteria plated on zeocin/kanamycin double-selectionplates as above and incubated at 37° C.

The resolved BAC had lost the duplication of the 44 kb overlap and thesequence for pBeloBAC vector 3 (FIG. 5, right hand BAC map). E. colicolonies that grew on the double-selection plates exhibitedKm^(R)zeo^(R), were picked and BAC DNA isolated by miniprep. BAC DNA wasdigested with NotI and run on pulse-field gels. Clones exhibited theexpected pattern of bands (FIG. 5, right BAC map and right gel photo).The resolved BAC was 489 kb in total size of which 467 kb is humangenomic DNA from, in 5′ to 3′ order, Vκ2-30 through the 3′ cisregulatory regions, including 16 functional Vκ genes, the entire Jκcluster and Cκ.

Example 3 In Silico Assembly of the Sequence of a Functional 194 KbSynthetic Human Ig Lambda Light Chain Transgene

The complete annotated sequence of the human immunoglobulin lambda lightchain locus (Igλ) is available. For example see GenBank(http://www.ncbi.nlm.nih.gov/genbank/) Accession Number NG_000002.Additional detailed information, including bibliographic supportingscientific references is available at several public domain websitesincluding Vbase (http://vbase.mrc-cpe.cam.ac.uk/) and IMGT(http://imgt.cines.fr/). This information includes data for the geneticand phenotypic content of the human Igλ locus, for instance including,but not limited to, identification of expressed gene sequences,pseudogenes, allelic variants, and which genes may encode domains proneto misfolding.

Using such public information, it is possible to assemble DNA sequencesin silico using commonly available software for manipulating DNAsequences (e.g., MacVector, DNASIS) that encode a human Igλ light locuscomprising only expressed human Vλ genes in operational linkage withfrom one to all 7 human Jλ-Cλ pairs and the complete functional humanIgλ 3′ enhancer (3′ E). Cis regulatory elements controlling Vλ geneexpression may be captured on as little as 500 bp of DNA immediately 5′to the start of the 5′ untranslated region (UTR) and 500 bp or less DNAimmediately 3′ to the recombination signal sequence (RSS) immediately 3′of the end of the coding sequence of each Vλ gene. Preferably, a largerregion of DNA 5′ of the start of the 5′ UTR may be used to increase thedistance between V gene segments and to capture fully any and all cisregulatory elements.

Furthermore, the region between the most 3′ of the human Vλ genes (V3-1)and Jλ1-Cλ1, the first Jλ-Cλ pair, and through the 3′ E is captured ingermline configuration. Alternatively, the distance of the sequencebetween Vλ3-1 and Jλ1-Cλ1 and/or the distance between Jλ7-Cλ7, the lastJλ-Cλ pair in the human locus, and the 3′ E may be truncated. Thespecific distances are not so important as capturing of desired codingelements and critical cis regulatory elements including splice acceptorsand splice donors, RSSs, intronic enhancer and 3′ enhancer, preferablyall 3 DNAseI hypersensitive sites. Furthermore, the human Igλpseudogenes, Jλ4-Cλ4, Jλ5-Cλ5 and/or Jλ6-Cλ6 may be excluded from the insilico assembled sequence. There is a de minimus requirement for onefunctional Jλ-Cλ pair, either Jλ1-Cλ1, Jλ2-Cλ2, Jλ3-Cλ3 or Jλ7-Cλ7.

Specific restriction enzyme sites may be introduced at the end of thesequence. Specific restriction enzymes sites also may be introduced ordeleted internally through sequence insertion, deletion or modificationso long as they do not perturb gene expression or coding. These enzymessites may include sequences useful for assembling the synthesizedsequence in vitro, excising the DNA from the vector or for variousscreening methodologies, such as Southern blot of agarose gel usingstandard electrophoresis, field inversion gel electrophoresis (FIGE),and pulsed-field gel electrophoresis (PFGE).

Inserted sequences may also include primer binding sites to facilitatePCR-based screening methods including qPCR, for the desired and intactintegration into the genome. Optionally, a site-specific recombinasesite(s) such as loxP or any of its variants or frt are introduced tofacilitate deletion of intervening sequences to make a single-copytransgene, to facilitate introduction of additional DNA via sitespecific recombination, or to facilitate other genetic engineeringdesigns as known in the art. Also optionally included may be sequencesfor drug-selection cassettes for mammalian cells such as a positiveselection marker for resistance to a drug such as hygromycin or anegative selection cassette such as thymidine kinase.

Using the strategy outlined above, a core 191 kilobase sequence (“LambdaPrime”) was assembled in silico, comprising 29 functional human Vλ geneson approximately ˜5 kb units, all 7 human Jλ-Cλ pairs and the human 3′Igλ locus enhancer, with the 57 kilobase sequence between Vλ3-1 and thehuman 3′ Enhancer in germline configuration. The 29 chosen human Vλgenes were documented to be expressed in humans and present in all knownhuman haplotypes as determined by investigation of the scientificliterature. Sequences for the most commonly used alleles that encodevariable regions that fold properly were chosen.

In instances in which two functional Vλ genes were positioned inproximity of less than 5 kb distance in the human germline configurationthe entire sequence comprising the two Vλ genes, from approximately 4 kb5′ of the 5′ UTR of the most 5′ gene and approximately 500 bp 3′ of theRSS of the most 3′ Vλ gene, was used. The coding and non-coding regionsof the sequence were sufficient to drive proper developmental regulationand expression and to generate a diversity of human Igλ light chainsonce introduced into the mouse genome.

A sequence for a hygromycin resistance expression cassette was inserted5′ of the most 5′ Vλ cassette. For ease of excision from the BAC vectorand for confirming intact integration, rare cutting restriction enzymeswere inserted into the sequence, at the 5′ end, recognition sequencesfor StuI/EcoRV/AsiSI/PvuI and AgeI/PacI/AseI/BsaBI sites 5′ and 3′ ofthe hyg^(R) cassette, respectively, and at the 3′ end downstream of the3′ enhancer, recognition sequences for AsiSI, AleI, EcoRI, Bsa BI wereinserted.

Example 4 In Silico Assembly of the Sequence of Two Synthetic Human IgLambda Light Chain Transgenes Derived from the Sequence of IgLambda-Prime

Using the Lambda-Prime sequence described in Example 3, two additionaltransgenes were designed. A core 94 kilobase sequence (“Lambda 3”) wasassembled in silico, comprising 8 functional human Vλ genes onapproximately ˜5 kb units, all 7 human Jλ-Cλ pairs and the human 3′ Igλlocus enhancer, with the 57 kilobase sequence between Vλ3-1 and thehuman 3′ Enhancer in germline configuration. The 8 chosen human Vλ geneswere documented to be expressed in humans. Sequences for the mostcommonly used alleles that encode variable regions that fold properlywere chosen. The coding and non-coding regions of the sequence weresufficient to drive proper developmental regulation and expression andto generate a diversity of human Igλ light chains once introduced intothe mouse genome. An frt site was inserted 5′ of the most 5′ Vλ genecassette. For ease of excision from the BAC vector and for confirmingintact integration, rare cutting restriction enzymes were inserted intothe sequence, at the 5′ end, recognition sequences for ApaLI/AvrII/EcoRIsites were inserted 5′ of the frt site and a recognitions sequence forFseI was inserted 3′ of the frt site and, at the 3′ end, the recognitionsequences for AsiSI, AleI, EcoRI, Bsa BI described in Example 3 wereretained.

Using the Lambda-Prime sequence described in Example 3, a sequence(“Lambda 5”) was assembled in silico comprising 21 human Vλ genes withdemonstrated expression and functionality, and with no knownnon-functional alleles or haplotypic variation across individual humans.The Vλ cassettes were generally approximately 5 kb in size. The codingand non-coding regions of the sequence were sufficient to drive properdevelopment regulation and expression and to generate a diversity ofhuman Igλ light chains once introduced into the mouse genome inoperational linkage with any DNA construct comprising at least onefunctional Jλ-Cλ pair and preferably a functional 3′ E. Sequences forthe most commonly used alleles that encode variable regions that foldproperly were chosen.

A sequence for a hygromycin resistance expression cassette 5′ of themost 5′ Vλ cassette as described in Example 3 was retained. The sequencefor an frt site was inserted 3′ of the most 3′ Vλ cassette. For ease ofexcision from the BAC vector and for confirming intact integration, rarecutting restriction enzymes were inserted into the sequence,StuI/EcoRV/AsiSI/PvuI sites 5′ of the hyg^(R) cassette,AgeI/PacI/AseI/BsaBI sites 3′ of the hyg^(R) cassette, as described inExample 3, and FseI/PvuI sites 5′ of the frt site and KpnI/NheI sites 3′of the frt site.

Example 5 Synthesis and Assembly of DNAs Comprising the Lambda 3 andLambda 5 Transgenes

DNAs of greater than approximately 30-40 kb in size are carried on BACs.Example 2 documents the creation of a BAC 545 kb in size. In addition,other cloning vectors capable of carrying large pieces of DNA such asYACs, PACs, MACs, may be used. Genetic engineering and physical recoveryof large DNAs in all of these vectors is well-documented in theliterature.

Contract service providers synthesize and assemble very large pieces ofDNA. The DNA sequence of Lambda 3 was transmitted to DNA2.0, Inc. (MenloPark, Calif.). The sequence was synthesized into physical DNA andassembled. The final fully assembled sequence was carried in a BAC withpBeloBAC as the vector backbone. The full BAC was sequenced bySeqWright, Inc. (Houston, Tex.) using 454 sequencing technology (454Life Sciences, Roche). The sequence of the synthetic Lambda 3 DNA wasconfirmed against the reference sequence. Six sequence deviations fromthe in silico sequence were likely 454 sequencing read errors due tolong homopolymeric or dipolymeric sequences. The deviations, even thoughvery likely not mutations in the actual physical synthetic DNA, mappedto non-coding, non-regulatory regions.

The DNA sequence of Lambda 5 was transmitted to DNA2.0, Inc. (MenloPark, Calif.). The sequence was synthesized and assembled. The finalfully assembled sequence was carried in a BAC with pBeloBAC as thevector backbone. The full BAC was sequenced by SeqWright, Inc. (Houston,Tex.) using 454 sequencing technology (454 Life Sciences, Roche). Thesequence of the synthetic Lambda 5 DNA was confirmed against thereference sequence. Minimal deviations from the in silico sequence werefound. Any deviations were likely 454 sequencing read errors due to longhomopolymeric or dipolymeric sequences. The deviations, even though verylikely not mutations in the actual physical synthetic DNA, map tonon-coding, non-regulatory regions.

Example 6 Generation of Transgenic Mice Carrying the Synthetic Lambda 3Transgene

The Lambda 3 BAC was digested with FseI and AsiSI and the synthetichuman Lambda 3 insert purified from the vector sequence by pulse-fieldgel electrophoresis. The 94 kb gel band containing the Lambda 3 sequencewas excised from the gel and purified from the gel. The purified,concentrated DNA was microinjected into the pronucleus of fertilizedmouse eggs. Of 758 embryos transferred, 138 live mice were born. PCRassays to detect human Igλ sequence comprising the 5′ and 3′ ends and inthe middle of the Lambda 3 transgene were used to screen DNAs isolatedfrom tail tissue from mouse pups to screen for the presence of DNA atthe 5′, 3′ and the middle of the Lambda 3 transgene. Twenty-four mousepups were confirmed positive for all three PCR products. ELISA specificfor human Igλ was performed on serum samples from the founder mice.Twenty independent founder mice were found to have significantcirculating levels of human Igλ in their serum, confirming function ofthe Lambda 3 transgene. Founder mice were bred to produce transgenicoffspring.

Example 7 Expression of a Diversity of Human Ig Lambda Light Chains fromthe Synthetic Human Lambda 3 Transgene in Mice

Samples of serum from the transgenic offspring of the founder mice areconfirmed to have the intact and expressed Lambda 3 transgene asdescribed in Example 6.

Blood is drawn from Lambda 3 transgenic mice and collected inheparinized tubes. Lymphocytes are separated and concentrated viadensity gradient centrifugation over Lympholyte M. The lymphocytes aretreated with fluorochrome-conjugated antibodies against a mouse B cellmarker, e.g., B220 or CD19, and an antibody specific for human Igλ.Mouse B cells expressing human Igλ light chains on their surface aredetected by FACs. The percentage of human Igλ positive B cells rangesfrom 1 to 40% or more.

mRNA is isolated from lymphoid tissue, e.g., spleen, lymph nodes, bonemarrow, blood, of the Lambda 3 transgenic mice and RT-PCR using primersspecific for human Vλ and Cλ is used to amplify the expressed repertoireof human variable regions from the Lambda 3 transgene. The Vλ cDNAs arecloned into a cloning vector such as TA (Invitrogen, Inc., Carlsbad,Calif.). The human Vλ cDNAs are sequenced. All 8 Vλ genes and thefunctional human Jλ-Cλ are shown to be represented in the expressedrepertoire. The sequence of the cDNAs have an open-reading frame andencode fully human variable regions, consistent with functionalrecombination of the Vλ-Jλ and appropriate development regulation of thehuman Igλ transgene.

Mice transgenic for Lambda 3 are immunized with antigen using methodsknown in the art. mRNA is isolated from the secondary lymphoid tissue,e.g., spleen, lymph nodes, of the Lambda 3 transgenic mice and RT-PCRusing primers specific for human Vλ and Cλ is used to amplify theexpressed repertoire of human variable regions from the Lambda 3transgene. The Vλ cDNAs are cloned into a cloning vector such as TA. Thehuman Vλ cDNAs are sequenced. The human Vλ regions are found to bemutated as compared to the germline sequence, indicative of somaticmutation events consistent with affinity maturation.

Taken together these data demonstrate that the Lambda 3 transgene isexpressed in B cells, expresses a diversity of human Igλ light chains,and is a template for somatic mutation events indicative of itundergoing affinity maturation in the secondary immune response.

Example 8 Generation of Transgenic Mice Carrying a 194 Kb SyntheticHuman Ig Lambda Transgene, Lambda-Prime, by Pronuclear Co-Microinjection

The synthetic, sequence confirmed Lambda 5 BAC is digested with AsiSIand FseI, run on an agarose gel in PFGE and DNA comprising the Lambda 5sequence is isolated as in Example 6. This DNA is co-microinjected withDNA comprising the Lambda 3 sequence, isolated as in Example 6, into thepro-nucleus of fertilized mouse eggs. The co-microinjected DNAco-integrates into the mouse genome, with a significant proportion ofthe integration events comprising Lambda 5 and Lambda 3 oriented inoperable linkage, i.e., both are oriented in the same 5′ to 3′orientation respective to each other and Lambda 5 is integrated 5′ toLambda 3, i.e., the 3′ end of Lambda 5 is juxtaposed to the 5′ end ofLambda 3. Thus, the contiguous human sequence of Lambda-Prime, 194 kb ofsynthetic DNA in operable linkage is created, comprising 29 functionalhuman Vλ sequences, all human Jλ-Cλ and the human 3′ enhancer sequence.Intact integration in operably linkage is confirmed by Southern blots ofgenomic DNAs cut with rare cutting restriction enzymes, run on standardand PGFE gels and probed with sequences specific to Lambda 5 and Lambda3. Because the full nucleotide sequence of an operably-linkedco-integrated Lambda-Prime sequence is fully known, in silico predictionof restriction fragment patterns is readily accomplished to confirmintact and operable linkage, as facilitated by the rare-cuttingrestriction enzyme sites designed into the sequences as outlined inExample 4. Transgene function is confirmed by ELISA for human Igλ in theserum.

Founder mice are bred and transgenic offspring are produced. Copy numberis readily assessed by methods such as qPCR or densitometric scanning ofSouthern blots of genomic DNA. If desired, in lines in which multi-copyLambda 5-3 transgenes are integrated, transgenic mice are bred totransgenic mice expressing FLP-recombinase. The frt sites present in theLambda 5 and Lambda 3 transgenes recombine site-specifically, particularin the germline cells. Gametes are produced that have a resolvedsingle-copy transgene of Lambda 5-Lambda 3 operably linked and thesegametes transmit the single-copy resolved Lambda-Prime sequence into thenext generation.

Transgenic mice, either multicopy or single copy, are tested for LambdaPrime function as described in Example 7. The data demonstrate that theLambda-Prime transgene is expressed in B cells, expresses a diversity ofhuman Igλ light chains, and is a template for somatic mutation eventsindicative of it undergoing affinity maturation in the secondary immuneresponse.

Example 9 Generation of Transgenic Mice Carrying a 194 Kb SyntheticHuman Ig Lambda Transgene by Co-Transfection into ES Cells

DNAs comprising the Lambda 3 and the Lambda 5 sequences are isolated asin Example 8. These DNAs are co-introduced into mouse ES cells by amethod such as lipofection or electroporation. The presence of apositive-selectable maker cassette 5′ of the most 5′ Vλ gene on Lambda5, e.g., hygromycin, enables positive selection for integration ofLambda 5. The co-introduced DNA randomly co-integrates into the mousegenome, with a significant proportion of the integration eventscomprising Lambda 5 and Lambda 3 oriented in operable linkage, i.e.,both are oriented in the same 5′ to 3′ orientation respective to eachother and Lambda 5 is integrated 5′ to Lambda 3′, i.e., the 3′ end ofLambda 5 is juxtaposed to the 5′ end of Lambda 3. Thus, the contiguousLambda-Prime sequence of 194 kb of synthetic DNA in operable linkage iscreated.

Intact integration in operably linkage is confirmed by Southern blots ofgenomic DNAs cut with rare cutting restriction enzymes, run on standardand PGFE gels and probed with sequences specific to Lambda 5 and Lambda3. Because the full nucleotide sequence of an operably-linkedco-integrated Lambda-Prime sequence is fully known, in silico predictionof restriction fragment patterns is readily accomplished to confirmintact and operable linkage, as facilitated by the rare-cuttingrestriction enzyme sites designed into the sequences as outlined inExample 4. Copy number may be readily assessed by methods such as qPCRor densitometric scanning of Southern blots of genomic DNA. If desired,in clones in which multi-copy Lambda 5-3 transgenes are integrated,FLP-recombinase is transiently expressed in the clones. The frt sitespresent in the Lambda 5 and Lambda 3 transgenes recombinesite-specifically. Clones are produced that have a resolved single-copytransgene of Lambda 5-Lambda 3 operably linked.

ES cells carrying the operably linked Lambda-Prime transgene sequenceare used to generate transgenic mice using well-established methods. Forexamples, ES cells are microinjected into mouse blastocysts, which arethen implanted in pseudo-pregnant foster females. Chimeric pups areborn. Chimeric mice are bred and the resulting offspring are screenedfor the presence of the Lambda-Prime transgene.

Transgenic mice, either multicopy or single copy, are tested forLambda-Prime function as described in Example 7. The data demonstratethat the Lambda-Prime transgene is expressed in B cells, expresses adiversity of human Igk light chains, and is a template for somaticmutation events indicative of it undergoing affinity maturation in thesecondary immune response.

Example 10 Synthesis and Assembly of a DNA Comprising the Lambda-PrimeTransgene and Generation of Transgenic Mice Therefrom

DNAs of greater than approximately 30-40 kb in size are carried on BACs.Example 2 documents the creation of a BAC 545 kb in size. In addition,other cloning vectors capable of carrying large pieces of DNA such asYACs, PACs, MACs, may be used. Genetic engineering and physical recoveryof large DNAs in all of these vectors is well-documented in theliterature.

Contract service providers synthesize and assemble very large pieces ofDNA. The DNA sequence of Lambda-Prime is transmitted to one searchservice provider, DNA2.0, Inc. (Menlo Park, Calif.). The sequence issynthesized into physical DNA and assembled. The final fully assembledsequence is carried in a BAC with pBeloBAC as the vector backbone. Thefull BAC was sequenced by sequencing service providers such asSeqWright, Inc. (Houston, Tex.) using 454 sequencing technology (454Life Sciences, Roche) or standard shotgun sequencing. The sequence ofthe synthetic Lambda-Prime DNA is confirmed against the referencesequence. Any sequence deviations from the in silico sequence are likelysequencing read errors due to long homopolymeric or dipolymericsequences. The deviations, even though very likely not mutations in theactual physical synthetic DNA, map to non-coding, non-regulatoryregions.

Alternatively, Lambda 3 and Lambda 5 may be recombined in vitro usingtechniques as described in Examples 1 and 2. They may also be recombinedusing other methods of engineering BACs such as recombineering, orstandard restriction fragment ligation into pBeloBAC following bytransfection into E. coli.

Transgenic mice are generated as described in Examples 6 or byintroduction into ES cells such as by electroporation, lipofection etc.,as exemplified in Example 9. Transgenic mice, either multicopy or singlecopy, are tested for Lambda-Prime function as described in Example 8.The data demonstrate that the Lambda-Prime transgene is expressed in Bcells, expresses a diversity of human Igλ light chains, and is atemplate for somatic mutation events indicative of it undergoingaffinity maturation in the secondary immune response.

Example 11 Creation of a Human Igl Transgene Via Co-Introduction ofLambda 3 with Genomic DNA Comprising Additional Human V LambdaRepertoire

Libraries of human genomic DNA are available commercially or throughlicensing and are well-characterized. These include the CalTech humangenomic library carried on BACs and various human genomic DNA librarieson YACs. The CalTech human library BAC clones from libraries B, C and Dmay be ordered through Invitrogen (Carlsbad, Calif.). Human genomiclibraries are also available carried on cosmids, phage, P1s, PACs etc.All of these vectors may be modified prior to co-introduction usingtechniques readily available in the art.

Because of the ready facility by which large fragments of DNA may besequenced, the genomic inserts on these BACs or YACs may be sequencedconfirmed using a contract service provider as described above. Thecomplete human DNA insert may be isolated. Alternatively, a subfragmentmay be isolated using rare-cutting restriction enzyme sites available inthe genomic DNA. An example of a suitable YAC is L1 (U.S. Pat. No.7,435,871). Other YAC, BAC and cosmid clones suitable for use aredescribed in Kawasaki et al., (Gen. Res. (1995) 5: 125-135) and Frippiatet al. (Hum. Mol. Genet. (1995) 4: 983-991). One or more BACs or YACscomprising additional human Vλ genes are co-introduced with the Lambda 3construct. Optionally, the Lambda 3 DNA is co-introduced with two ormore other constructs with additional Vλ genes. The two or moreco-introduced constructs are confirmed to co-integrate in operablelinkage as outlined in Examples 8 and 9. Transgene functionality isconfirmed as in Example 7. Thus, a human Igλ transgene may be partlysynthetic and partly derived from a genomic library, with the core Jλ-Cλand 3′ cis regulatory sequences created by synthetic means and all orpart of the Vλ repertoire derived from a genomic library.

Example 12 Creation of a Human Igl Transgene Via Co-Introduction ofLambda 5 with a Genomic DNA Sequence Comprising at Least One FunctionalHuman JL-CL Pair

Libraries of human genomic DNA are available commercially or throughlicensing and are well-characterized. These include the CalTech humangenomic library carried on BACs and various human genomic DNA librarieson YACs. The CalTech human library BAC clones from libraries B, C and Dmay be ordered through Invitrogen (Carlsbad, Calif.). Human genomiclibraries are also available carried on cosmids, phage, P1s, PACs etc.All of these vectors may be modified prior to co-introduction usingtechniques readily available in the art.

Because of the ready facility by which large fragments of DNA may besequenced, the genomic inserts on these BACs or YACs may be sequencedconfirmed using a contract service provider as described above. Thecomplete human DNA insert may be isolated. Alternatively, a subfragmentmay be isolated using rare-cutting restriction enzyme sites available inthe genomic DNA. An example of a suitable YAC is L2 (U.S. Pat. No.7,435,871), which contains all 7 Jλ-Cλ pairs and the human 3′ enhancer.Other YAC, BAC and cosmid clones suitable for use are described inKawasaki et al., (Gen. Res. (1995) 5: 125-135) and Frippiat et al. (Hum.Mol. Genet. (1995) 4: 983-991).

The core construct contains at least one functional human Jλ-Cλ pair andpreferably a functional 3′ enhancer. The Lambda 5 DNA is co-introducedwith the isolated DNA of the core construct and, optionally, one or moreother constructs with additional Vλ genes. The two or more co-introducedconstructs are confirmed to co-integrate in operable linkage as outlinedin Examples 8 and 8. Transgene functionality is confirmed as in Example7. Thus, a human Igλ transgene may be partly synthetic and partlyderived from a genomic library, with the core Jλ-Cλ and 3′ cisregulatory sequences derived from a genomic library and all or part ofthe Vλ repertoire created by synthetic means.

Example 13 Use of Cre-Lox System to Recombine Transgenes

The sequence of the Lambda 3 transgene is designed in silico asdescribed in Example 4 with the alteration of an addition of a loxP siteor variant thereof replacing the sequence of the frt site or being placeadjacent to it, and a drug-resistance cassette activity in mammaliancells such as puromycin-resistance is inserted 5′ to the loxP site,creating Lambda 3P. The Lambda 3P sequence is synthesized and assembledinto physical DNA as described in Example 5. The Lambda 3P DNA isisolated from the vector DNA, introduced into ES cells,puromycin-resistance colonies selected for, picked and molecularlyscreened for intact integration of Lambda 3P.

The sequence of the Lambda 5 transgene is designed in silico asdescribed in Example 4 with the alteration of an addition of a loxP siteor variant thereof replacing the sequence of the frt site or being placeadjacent to it, creating Lambda 3P. The Lambda 5P sequence issynthesized and assembled into physical DNA as described in Example 5except that the BAC vector sequence, such as pBeloBAC, has a deletedloxP site or carries a version incompatible for recombination with thatin the Lambda 5P sequence. The circular Lambda 5P BAC DNA is isolatedand co-transfected with CRE recombinase into Lambda 3P ES clones. TheCRE recombinase engenders site-specific recombination between the loxPsites, resulting in integration of the Lambda 5P DNA in operably linkageupstream of the Lambda 3 DNA, therein reconstituting the Lambda Primesequence. Lambda 5P positive ES clones are selected forpuromycin-resistance, picked and molecularly screened for insertion ofLambda 5P into Lambda 3P as described in Example 9. Transgenic mice aregenerated from the ES cells and confirmed for Lambda Prime transgenefunction as described in Example 9.

This process for insertion of additional Vλ repertoire upstream of afunctional core Jλ-Cλ sequence is applicable for any vector existing asa circular DNA, e.g., plasmid, cosmid, BAC, or circularizable, such as aYAC, so long as the loxP site is 3′ of most 3′ Vλ gene desired to beoperably linked to the Jλ-Cλ core sequence.

Example 15 Generation of Mice Transgenic Expressing Human Ig Lambda froma Synthetic DNA Transgene Comprising a Highly Chimeric Human-Mouse DNASequence

The annotated sequence of the mouse immunoglobulin lambda light chainlocus is available in the public domain, see Genbank accession numberNC_000082. Because of the unique structure of the mouse Igλ locus, whichis composed of two separates units (see Selsing et al., ImmunoglobulinGenes 1989 Acad. Press Ltd., pp. 111-122), the sequence of one of themouse Igλ locus units is selected. A 60,000 nucleotide (nt) sequencecomprising 4 kb upstream of the start codon of Vλ1 and 5 kb downstreamof the 3′ enhancer is isolated in silico. A sub-sequence of 4 kbupstream of the start codon of mouse Vλ2 and 500 bp downstream of theRSS is identified (“Vλ expression cassette”). FIG. 1 of Ramsden and Wu(Proc. Natl. Acad. Sci. 1991 88: 10721-10725) identifies the Vλ2 RSS andthe RSS for Jλ3 and Jλ1. The sequence of the 39 nucleotide RSS of Vλ1 ofmouse is replaced in the functional orientation with the functional RSSfrom a human Vλ, e.g., Vλ3-1. This approximately 5,000 nucleotidesequence comprising 4,000 nt upstream of the start codon, human RSS andthrough 500 nt downstream of the RSS, is the core Vλ expressionconstruct.

The 28 nucleotide sequence of mouse Jλ3 and Jλ1 are replaced in thefunctional orientation with the functional RSS from human Jλ3 and Jλ1.The coding sequences for mouse Jλ3 and Jλ1 are replaced by the codingsequences for human Jλ3 and Jλ1. The coding sequences for mouse Cλ3 andCλ1 are replaced by the coding sequences for human Cλ3 and Cλ1. Thecoding sequence of mouse Vλ1 is replaced with the coding sequence of ahuman Vλ gene, e.g., Vλ3-1. The sequence comprising the mouse 3′enhancer is replaced with 7,562 nucleotide sequence comprising the 3DNAseI hypersensitive sites of the human Igλ 3′ enhancer. This sequenceis the core chimeric Igl construct. Combriato and Klobeck (J. Immunol.2002 168:1259-1266) teach other sequence changes for restoring optimalenhancer activity to the mouse 3′ enhancer.

Additional Vλ repertoire is added in silico through appending the coreVλ expression construct sequence 5′ to the core chimeric Igλ construct.In each appended Vλ expression construct, the mouse Vλ1 coding sequenceis replaced with human Vλ coding sequence. The entire human Vλrepertoire can be appended sequentially in silico yielding a sequence ofapproximately 205,000 nt.

The sequence or two portions thereof is synthesized and assembled intophysical DNA is described in previous examples. The DNA is used toconstruct transgenic mice as described in previous examples. Transgenicmice are analyzed for transgene expression and function as described inprevious examples. The data demonstrate that the transgene is expressedin B cells, expresses a diversity of human Igλ light chains, and is atemplate for somatic mutation events indicative of it undergoingaffinity maturation in the secondary immune response.

The preceding example illustrates the methodology by which exquisitelyprecisely and complexly engineered sequences are composed in silico andthen a process for making transgenic animals comprising that sequence.The methodology is not limited to the described sequence.

Example 14 In Silico Assembly of the Sequence of a Functional SyntheticHuman Ig Kappa Light Chain Transgene

The methodologies described in the preceding examples are broadlyapplicable for the in silico assembly and subsequent synthesis of anysequence up to the cloning capacity of a BAC, which as demonstrated inExample 2, is at least 500 kb. As described in Example 3, a sequenceencoding a human Igκ transgene was assembled from publicly availableinformation on the sequence of the human and mouse Igκ loci. Theannotated sequence for the complete human Igκ locus was accessed fromGenbank, accession number NG_000834. The sequence comprises the completeproximal Vκ cluster through the 3′ regulatory elements, 3′ enhancer, Edand RS. Additional detailed information, including bibliographicsupporting scientific references is available at several public domainwebsites including Vbase (http://vbase.mrc-cpe.cam.ac.uk/) and IMGT(http://imgt.cines.fr/). This information includes data for the geneticand phenotypic content of the human Igκ locus, for instance including,but not limited to, identification of expressed gene sequences,pseudogenes, allelic variants, and which genes may encode domains proneto misfolding.

A 30,000 nt sequence comprising 4 kb upstream of human Vκ4-1 through thecomplete human Jκ cluster through 1,000 nt 3′ of human Cκ was ingermline configuration. Appended in silico 3′ of human Cκ was a 25,600nt germline configured mouse DNA sequence comprising the Igκ 3′enhancer, Ed and RS. This sequence served as the core Igκ expressioncassette.

To expand the repertoire, sequence for additional Vκ expressioncassettes as units of ˜5,000 nt were added 5′ of Vκ4-1. In instances inwhich two functional Vκ genes were positioned in proximity of less than5 kb distance in the human germline configuration the entire sequencecomprising the two Vκ genes, from approximately 4 kb 5′ of the 5′ UTR ofthe most 5′ gene and approximately 500 bp 3′ of the RSS of the most 3′Vκ gene, was used. As described in Example 3, recognition sequences forspecific restriction enzymes were introduced at the ends of thesequence. Recognition sequences for specific restriction enzymes wereintroduced and deleted internally through sequence insertion, deletionor modification; these did not perturb gene expression or coding.

Example 16 Generation of Mice Transgenic for a Locus Expressing HumanIgk, Said Locus Comprising Synthetic DNA

Using methodology described in any of the preceding Examples 1-2 andExamples 4-14 and the process for in silico assembly of sequencesdescribed in Examples 3 and 15, physical DNA that encodes human Igκlight chains is synthesized and used to create transgenic mice.Transgenic mice are analyzed for transgene expression and function asdescribed in previous examples using appropriate reagents for use instudying human Igκ expression at the nucleic acid and protein levels.The data demonstrate that the transgene is expressed in B cells,expresses a diversity of human Igκ light chains, and is a template forsomatic mutation events indicative of it undergoing affinity maturationin the secondary immune response.

Example 17 Generation of Mice Expressing Human Igk from a Synthetic DNATransgene Comprising a Highly Chimeric Human-Mouse DNA Sequence

The annotated sequence of the human immunoglobulin kappa light chainlocus is publicly available, see Genbank accession number NG_000834. Theannotated sequence of the mouse immunoglobulin kappa light chain locusis publicly available, see Genbank accession number NC_005612. Othersources such as the IGMT Repertoire website (http://imgt.cines.fr/) areused as a resource on the map and functionality of individual componentsin the loci. A mouse DNA sequence of approximately 50,000 bases,comprising a Vκ, preferably the most proximal mouse Vκ, Vκ3-1, the Jκcluster, Cκ through 3′ regulatory regions, is isolated in silico. Thoughthis sequence is preferably in germline configuration, intergenicregions of DNA may be deleted to make a smaller overall sequence so longas critical regulatory regions such as the Igκ intronic enhancer, 3′enhancer, Ed and RS, the sequence and location of which are alldocumented publicly, are unpertubed.

The mouse exons for the Vκ, Jκ and Cκ are replaced with their humancounterparts. The human Vκ exons replacing the mouse Vκ exons may beVκ4-1, which is human Vκ most proximal to the human Jκ cluster but thisis not absolutely necessary. Any human Vκ exon sequences may be used. Itis noted that human Vκ4-1 and the next most proximal Vκ gene, Vκ5-2, areinverted 3′-5′ relative to the human Jκ cluster in the germlineconfiguration. The human Vκ 4-1 exons would be oriented in the mouse Vκcontext in the 5′-3′ orientation relative to the mouse Jκ locus in thesequence constructed in silico. The mouse Jκ locus comprises 5 Jκsequences but Jκ3 may not be expressed because of a non-canonical donorsplice sequence. The human Jκ locus comprises 5 Jκ sequences, all ofwhich are functional. Incorporation of the human Jκ3 exon would bringwith it the proper splice donor sequence, particular for splicing to itscounterpart splice acceptor sequence on human Cκ.

Additional Vκ repertoire is added in silico through identifyingapproximately 5 kb units comprising in proximal to distal order thefunctional mouse Vκ genes. This number of 5 kb sequence units isequivalent to the number of human Vκ genes to be represented in thetransgenes. Mouse pseudogenes are eliminated. In each appended Vκexpression construct, the mouse Vκ coding sequence is replaced withhuman Vκ coding sequence. The 5 kb unit may also be a repeated unit sothat identical non-coding sequences comprise each unit and the units areonly distinguished by the unique human Vκ exon sequence. Each unit isappended onto the core sequence 5′ to the preceding one, sequentiallybuilding the sequence of the artificial locus, proximally to distally.

The entire proximal human Vκ repertoire can be appended sequentially insilico yielding a sequence of approximately 140,000 bases. The inverteddistal cluster of human Vκ genes may also be included, though becausethey are duplications of the genes in the proximal cluster, theycontribute to <10% of the expressed human Igκ repertoire, and becausethey are missing in some human haplotypes, their inclusion is notnecessary and may be undesired for later antibody drug development.

The sequence or two portions thereof is synthesized and assembled intophysical DNA is described in previous examples. The DNA is used toconstruct transgenic mice as described in previous examples. Transgenicmice are analyzed for transgene expression and function as described inprevious examples. The data demonstrate that the transgene is expressedin B cells, expresses a diversity of human Igκ light chains, and is atemplate for somatic mutation events indicative of it undergoingaffinity maturation in the secondary immune response.

The preceding example illustrates the methodology by which exquisitelyprecisely and complexly engineered sequences are composed in silico andthen a process for making transgenic animals comprising that sequence.The methodology is not limited to the described sequence.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

The invention claimed is:
 1. A method of producing an antibody, orantigen-binding fragment thereof, the antibody or fragment comprising ahuman immunoglobulin light chain variable region (VL) polypeptide,comprising: (a) immunizing a mouse with an antigen, wherein the mouse'sgenome comprises a transgene comprising (1) a plurality ofimmunoglobulin light chain variable (V) exons encoding humanimmunoglobulin light chain V polypeptides; (2) non-coding sequencesbetween the V exons; (3) a plurality of immunoglobulin light chainjoining (J) coding sequences encoding human immunoglobulin light chain Jpolypeptides; and (4) non-coding sequences between the J codingsequences; wherein the non-coding sequences between the V exons and thenon-coding sequences between the J coding sequences are derived frommouse immunoglobulin non-coding sequences, wherein the transgene iscapable of undergoing gene rearrangement to produce a polynucleotidesequence encoding the antibody or fragment comprising the immunoglobulinVL polypeptide; (b) recovering from the mouse a genomic DNA or cDNAcomprising a nucleotide sequence encoding the human immunoglobulin VLpolypeptide; and (c) recombinantly producing the human immunoglobulin VLpolypeptide.
 2. The method according to claim 1, wherein the non-codingsequences between the V exons and the non-coding sequences between the Jcoding sequences are derived from mouse immunoglobulin light chainnon-coding sequences.
 3. The method according to claim 1, wherein thenon-coding sequences between the V exons and the non-coding sequencesbetween the J coding sequences are selected from the group consisting ofintronic sequences and cis regulatory sequences.
 4. The method accordingto claim 3, wherein the cis regulatory sequences are selected from thegroup consisting of promoters, enhancers, recombination signalsequences, splice acceptor sequences, and splice donor sequences.
 5. Themethod according to claim 1, wherein the V exons encode human kappalight chain V (Vκ) polypeptides.
 6. The method according to claim 1,wherein the V exons encode human lambda light chain V (Vλ) polypeptides.7. The method according to claim 1, wherein the J coding sequencesencode human kappa light chain J (Jκ) polypeptides.
 8. The methodaccording to claim 1, wherein the J coding sequences encode human lambdalight chain J (Jλ) polypeptides.
 9. The method according to claim 1,wherein (1) the non-coding sequences between the V exons and (2) the Vexons are from non-orthologous immunoglobulin loci.
 10. The methodaccording to claim 1, wherein the transgene further comprises a codingsequence encoding an immunoglobulin light chain constant (CL)polypeptide.
 11. The method according to claim 10, wherein the codingsequence encoding the immunoglobulin CL polypeptide encodes a lambdalight chain constant (Cλ) polypeptide.
 12. The method according to claim10, wherein the coding sequence encoding the immunoglobulin CLpolypeptide encodes a kappa light chain constant (Cκ) polypeptide. 13.The method according to claim 10, wherein the immunoglobulin CLpolypeptide is a mouse immunoglobulin CL polypeptide.
 14. The methodaccording to claim 1, wherein the transgene further comprises mousenon-coding sequences upstream of the V exons.
 15. The method of claim14, wherein the non-coding sequences upstream of the V exons areselected from promoters and enhancers.
 16. The method according to claim1, wherein the transgene further comprises mouse non-coding sequencesdownstream of the J coding sequences.
 17. The method according to claim16, wherein the non-coding sequences downstream of the J codingsequences are selected from polyadenylation sites and 3′ untranslatedregions.
 18. The method according to claim 11, wherein the transgenecomprises (1) a plurality of human immunoglobulin light chain variableVλ exons encoding human immunoglobulin light chain variable Vλpolypeptides; (2) mouse non-coding sequences between the Vλ exons; (3)2-7 immunoglobulin light chain Jλ-Cλ coding sequence pairs, wherein eachJκ sequence encodes a human immunoglobulin light chain Jλ polypeptideand each Cλ sequence encodes a mouse immunoglobulin light chain Cλpolypeptide; (4) mouse non-coding sequences between the Jλ codingsequences and the Cλ coding sequences in the Jλ-Cλ coding sequencepairs; and (5) mouse non-coding sequences between the Jλ-Cλ codingsequence pairs.
 19. The method according to claim 12, wherein thetransgene comprises (1) a plurality of human immunoglobulin light chainvariable Vκ exons, encoding human immunoglobulin light chain variable Vκpolypeptides; (2) mouse non-coding sequences between the Vκ exons; (3) aplurality of human immunoglobulin light chain variable Jκ codingsequences encoding human immunoglobulin light chain variable Jκpolypeptides; (4) mouse non-coding sequences between the Jκ codingsequences; and (5) a Cκ coding sequence encoding a mouse immunoglobulinlight chain constant Cκ polypeptide.
 20. The method according to claim1, further comprising a step of appending the nucleotide sequenceencoding the human immunoglobulin VL polypeptide of step (b) to a DNAencoding a human light chain constant (CL) region to generate a DNAencoding a fully human VL-CL polypeptide.
 21. The method according toclaim 1, wherein the mouse's genome further comprises a second transgeneencoding an immunoglobulin heavy chain, or a portion thereof.
 22. Themethod according to claim 1, further comprising a step of isolating Blymphocytes from said mouse, wherein one or more of said B lymphocytesproduces an antigen-specific antibody that specifically binds to thetarget antigen.
 23. The method according to claim 22, further comprisinga step of screening said B lymphocytes to identify the one or more Blymphocytes that produces the antigen-specific antibody thatspecifically binds to the target antigen.
 24. The method according toclaim 23, wherein the recovering step (b) comprises isolating thegenomic DNA or cDNA comprising the nucleotide sequence encoding thehuman immunoglobulin VL polypeptide from the one or more B lymphocytesthat produces the antigen-specific antibody that specifically binds tothe target antigen.
 25. The method according to claim 22, wherein themethod further comprises generating hybridomas from the isolated Blymphocytes.
 26. The method according to claim 25, wherein therecovering step (b) comprises isolating the genomic DNA or cDNAcomprising the nucleotide sequence encoding the human immunoglobulin VLpolypeptide from the hybridoma that produces the antigen-specificantibody that specifically binds to the target antigen.
 27. The methodaccording to claim 22, further comprising implementing the nucleotidesequence encoding the human immunoglobulin VL polypeptide into an invitro antibody display system.
 28. The method according to claim 24,further comprising implementing the nucleotide sequence encoding thehuman immunoglobulin VL polypeptide into an in vitro antibody displaysystem.
 29. The method according to claim 26, further comprisingimplementing the nucleotide sequence encoding the human immunoglobulinVL polypeptide into an in vitro antibody display system.